FRET analyses of the U2AF complex localize
the U2AF35/U2AF65 interaction in vivo and
reveal a novel self-interaction of U2AF35
JANET CHUSAINOW,1,3PAUL M. AJUH,1LAURA TRINKLE-MULCAHY,1JUDITH E. SLEEMAN,1,4
JAN ELLENBERG,2and ANGUS I. LAMOND1
1Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom
2European Molecular Biology Laboratory, 69117 Heidelberg, Germany
We have analyzed the interaction between the U2AF subunits U2AF35 and U2AF65 in vivo using fluorescence resonance
energy transfer (FRET) microscopy. U2 snRNP Auxiliary Factor (U2AF) is an essential pre-mRNA splicing factor complex,
comprising 35-kDa (U2AF35) and 65-kDa (U2AF65) subunits. U2AF65 interacts directly with the polypyrimidine tract and
promotes binding of U2 snRNP to the pre-mRNA branchpoint, while U2AF35 associates with the conserved AG dinucleotide at
the 30end of the intron and has multiple functions in the splicing process. Using two different approaches for measuring FRET,
we have identified and spatially localized sites of direct interaction between U2AF35 and U2AF65 in vivo in live cell nuclei.
While U2AF is thought to function as a heterodimeric complex, the FRET data have also revealed a novel U2AF35 self-
interaction in vivo, which is confirmed in vitro using biochemical assays. These results suggest that the stoichiometry of the
U2AF complex may, at least in part, differ in vivo from the expected heterodimeric complex. The data show that FRET studies
offer a valuable approach for probing interactions between pre-mRNA splicing factors in vivo.
Keywords: pre-mRNA splicing; U2AF; fluorescence microscopy; fluorescence resonance energy transfer (FRET)
Pre-mRNA splicing is an essential step for the expression of
eukaryotic genes by which the noncoding segments from
precursor mRNA transcripts (pre-mRNAs) are specifically
removed and the coding sequences joined to form mature
mRNA. This process involves two sequential transesterifica-
tion reactions and is catalyzed by the spliceosome, a dynamic,
multicomponent complex. The spliceosome assembles in a
stepwise manner on the pre-mRNA from five smaller RNA–
protein subassemblies, called snRNPs (small nuclear ribonu-
cleoprotein particles), and numerous non-snRNP protein
splicing factors (for reviews, see Kra ¨mer 1996; Will and Luhr-
mann 1997; Staley and Guthrie 1998; Collins and Guthrie
2000). In two recent large-scale proteomic analyses of the
human spliceosome >300 distinct proteins have been identi-
fied (Rappsilber et al. 2002; Zhou et al. 2002; for reviews, see
Jurica and Moore 2003; Nilsen 2003).
Spliceosome assembly is directed by conserved pre-
mRNA sequences, including a short consensus sequence
at each exon–intron junction and a branch point (BP)
sequence followed by a polypyrimidine tract (Py tract),
located within the intron adjacent to the 30splice site (for
review, see Reed 2000). The BP contains an adenosine
residue that forms a phosphodiester bond with the 50end
of the intron during the first catalytic step of the splicing
reaction (Ruskin et al. 1984). Spliceosome assembly is
initiated by formation of the E complex, involving recogni-
tion of the 50splice site by U1 small nuclear RNP (Zhuang
and Weiner 1986; Seraphin and Rosbash 1989; Siliciano
and Guthrie 1988; Kohtz et al. 1994; Puig et al. 1999;
Zhang and Rosbash 1999; Labourier et al. 2001; Zhang
et al. 2001; Du and Rosbash 2002; Fo ¨rch et al. 2002),
binding of SF1/mBBP (splicing factor 1/branch-point bind-
ing protein) to the BP (Berglund et al. 1997; Rain et al.
1998), and U2 snRNP Auxiliary Factor (U2AF) to the Py
tract and the 30splice site (Zamore et al. 1992; Reed 1996,
2000; Moore 2000). In humans, the essential splicing factor
and Medical School, Dundee, DD1 9SY, United Kingdom.
Reprint requests to: Angus I. Lamond, Wellcome Trust Biocentre,
University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom;
e-mail: firstname.lastname@example.org; fax: +44-1382-345695.
Article and publication are at http://www.rnajournal.org/cgi/doi/10.1261/
3Bioprocessing Technology Institute, Singapore
4Division of Pathology and Neuroscience, Ninewells Hospital
RNA (2005), 11:1201–1214. Published by Cold Spring Harbor Laboratory Press. Copyright ª 2005 RNA Society.
U2AF, composed of 35-kDa (U2AF35) and 65-kDa
(U2AF65) subunits, is required for the recruitment of
U2 snRNP to pre-mRNAs. U2AF65 binds specifically to
the Py tract via its C-terminal RNA-binding domain,
which consists of three RNA recognition motifs (RRMs)
(Ruskin et al. 1988; Zamore et al. 1992; for review, see
Kra ¨mer 1996). U2AF65 stabilizes the interaction of U2
snRNP with the BP by contacting the branch region through
its N-terminal arginine- and serine-rich (RS) domain, pro-
moting base pair interactions between U2 snRNA and the BP
(Lee et al. 1993; Gaur et al. 1995; Valcarcel et al. 1996; Kent et
al. 2003). In previous models it was suggested that U2AF65
function is based on interactions between RRM3 and SF1/
mBBP, or the U2 snRNP component SAP155, respectively
(Berglund et al. 1997; Gozani et al. 1998). However, recent
observations indicate that RRM3 is dispensable for both
spliceosome assembly and splicing of certain introns in vitro
but plays an essential role in vivo (Banerjee et al. 2003, 2004).
Replacing SF1/mBBP, U2 snRNP associates with the BP
through base pairing interactions between the BP sequence
and U2 snRNA, while U2 snRNP proteins interact with
upstream sequences in the pre-mRNA to form the A complex
(Parker et al. 1987; Nelson and Green 1989; Wu and Manley
1989; Zhuang et al. 1989; Rosbash and Seraphin 1991; Gozani
et al. 1998; Staley and Guthrie 1998; Burge et al. 1999; Reed
In addition to its well-characterized role in constitutive
splicing, U2AF65 also plays an important role in regulated
splicing (Black 2000; Smith and Valcarcel 2000; Graveley
et al. 2001; Singh 2002). The U2AF35 binding domain of
U2AF65 is located in the proline-rich fragment between the
RS motif and RNA-binding region (Rudner et al. 1998;
Kielkopf et al. 2001). The small subunit U2AF35 contacts
the conserved AG dinucleotide at the 30splice site (Meren-
dino et al. 1999; Wu et al. 1999; Zorio and Blumenthal
1999). Its primary structure comprises a central RRM that
is flanked by two zinc fingers in the N terminus (Birney et al.
1993), and an RS domain and a glycine tract at the
C terminus (Zhang et al. 1992; Kellenberger et al. 2002).
U2AF35 binds both U2AF65 and the pre-mRNA through
its RRM domain. In addition to constitutive splicing,
U2AF35 plays an important role in the splicing of both
regulated introns and introns that have weak Py tracts
(Guth et al. 1999). Several studies support a model in
which splicing enhancer-bound SR proteins function by
recruiting U2AF65 to weak Py tracts (Bouck et al. 1998;
Wang et al. 1995; Zuo and Maniatis 1996; Graveley et al.
2001; for reviews, see Blencowe 2000 and Graveley 2000).
In this model, recruitment requires interactions between
the SR proteins and U2AF35. However, some studies are
inconsistent with the U2AF recruitment model (for
reviews, see Blencowe 2000 and Graveley 2000). Recent
experiments have demonstrated that, although the small
U2AF subunit is necessary for efficient enhancer-dependent
splicing, its RS domain is not required for this function
(Shepard et al. 2002). Both U2AF subunits have been shown
to bind to intronic sequences of pre-mRNA only during the
early steps of spliceosome assembly (Zamore et al. 1992;
Merendino et al. 1999; Wu et al. 1999; Zorio and Blu-
menthal 1999) and to be replaced later by the U5 snRNP
during B complex formation (Chiara et al. 1997).
To understand how the spliceosomal complex works, the
structures, functions, and interactions of its components
have to be determined. This includes the study of protein–
protein interactions between the various splicing factors. So
far, most of the work on analyzing direct functional inter-
actions between mammalian spliceosomal proteins has
been based on in vitro biochemical methods or yeast two-
hybrid systems. Here, we have used imaging techniques for
analyzing protein–protein interactions in the living cell to
complement in vitro techniques. To advance our work on
understanding functional interactions between essential
protein splicing factors in vivo, we have analyzed the inter-
action between U2AF35 and U2AF65 using fluorescence
resonance energy transfer (FRET) microscopy. FRET is a
powerful technique that can provide insight into the spatial
and temporal dynamics of protein–protein interactions in
vivo (for reviews, see Day et al. 2001; Wouters et al. 2001;
and Chen et al. 2003). Recently, Stanek and Neugebauer
(2004) have investigated the subnuclear distribution of
specific snRNP intermediates using a FRET assay. By ana-
lyzing distinct complexes containing the protein SART3,
which is required for U4/U6 snRNP assembly, they have
demonstrated that U4/U6 snRNP assembly occurs in Cajal
bodies. To localize the transient complex formed between
SART3 and the U4/U6 snRNP, SART3 and three U4/U6
snRNP-specific proteins were tagged with CFP or YFP, and
FRET was measured by acceptor photobleaching.
The U2AF subunits U2AF35 and U2AF65 bind tightly to
each other and form a stable complex, as previously demon-
strated by copurification and codepletion experiments, as well
as by cosedimentation on a glycerol gradient (Zamore and
Green 1989; Zhang et al. 1992; Guth et al. 1999). Using two
different microscopy systems and approaches for measuring
FRET, we have localized sites of direct interaction between
U2AF35 and U2AF65 in live cell nuclei. Interestingly, FRET
also revealed a novel U2AF35 self-interaction in vivo, which
was confirmed in vitro by using biochemical assays.
RESULTS AND DISCUSSION
U2AF35 and U2AF65 colocalize in nuclear
speckles with snRNP proteins and non-snRNP
protein splicing factors
For use in FRET analyses, EYFP (enhanced yellow fluores-
cent protein) and ECFP (enhanced cyan fluorescent pro-
tein) fusions of U2AF35 and U2AF65 were generated as
described in Materials and Methods. To compare their
subcellular localization with the localization patterns of
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Chusainow et al.
the corresponding endogenous proteins, the fusion proteins
were expressed in HeLa cells by transient transfection, and
the cells were fixed in 3.7% paraformaldehyde, immuno-
stained, and analyzed by fluorescence microscopy (Fig. 1).
The transiently expressed EYFP-U2AF35 fusion protein con-
centrates in speckles with a diffuse nucleoplasmic fraction
(Fig. 1A). Identical localization patterns were observed for
EYFP-U2AF65, as well as for ECFP fusion proteins of
U2AF35 and U2AF65 (data not shown; see Fig. 4). The
merged image in Figure 1C demonstrates the colocalization
of FP-tagged U2AF35 (Fig. 1A) with endogenous U2AF65
(Fig. 1B) in cells transiently expressing EYFP-U2AF35 and
immunostained with anti-U2AF65. Rabbit polyclonal anti-
peptide antibodies against U2AF35 did not recognize
U2AF35 when used for immunofluorescence cell staining.
Cells transiently coexpressing FP-U2AF35 and FP-U2AF65
also showed colocalization in nuclear speckles, in both fixed
and live cells (data not shown).
The localization patterns of U2AF35 and U2AF65 were
investigated further in stable cell lines constitutively expressing
either EYFP-U2AF35 (Fig. 1E) or EYFP-U2AF65 (Fig. 1I),
respectively. As in transiently expressing cells, the fusion pro-
teins are diffusely distributed in the nucleoplasm with addi-
tional concentration in nuclear speckles when constitutively
expressed in HeLa cells. HeLaEYFP-U2AF35cells (Fig. 1E) were
nizes the Sm family of snRNP proteins. HeLaEYFP-U2AF65cells
(Fig. 1I) were fixed and counterstained with an antibody
specific for the non-snRNP protein splicing factor SC-35
(Fig. 1J). The merged images shown in Figure 1, G and K,
respectively, demonstrate colocalization of EYFP-U2AF35
and EYFP-U2AF65 with both snRNP proteins and non-
snRNP protein splicing factors in nuclear speckles. The
U2AF proteins have a larger nucleoplasmic fraction than SC-
35 (Fig. 1I,J). These observations are in agreement with pre-
vious studies (Gama-Carvalho et al. 1997, 2001).
Characterization of stable cell lines
Stable cell lines constitutively expressing EYFP-U2AF35
(HeLaEYFP-U2AF35) and EYFP-U2AF65 (HeLaEYFP-U2AF65),
respectively, were generated as described in Materials and
Methods. In both cell lines, the FP-tagged protein is expressed
in >90% of cells and concentrates in speckles with a diffuse
nucleoplasmic fraction (Fig. 1E,I). This is identical to the
localization pattern observed with transiently expressing cells
(Fig. 1A; data not shown), as well as by immunostaining
endogenous protein in the parental HeLa cell line (shown for
U2AF65 in Fig. 1B) (Gama-Carvalho et al. 1997).
To analyze the expression levels of the FP-tagged proteins
in the stable cell lines, nuclear extracts (25 mg total protein)
from parental HeLa cells (Fig. 2A–C, lane 1) and stable
cell lines HeLaEGFP(Fig. 2A–C, lane 2), HeLaEYFP-U2AF35
(Fig. 2A–C, lane 3), and HeLaEYFP-U2AF65(Fig. 2A–C, lane
4) were separated by SDS-PAGE and probed on Western
blots with anti-U2AF35 (Fig. 2A), anti-U2AF65 (Fig. 2B),
and anti-GFP (Fig. 2C) antibodies. The anti-peptide anti-
body raised against U2AF35 for these studies (see Materials
and Methods) recognizes a single band of ?30 kDa for
endogenous U2AF35 (Fig. 2A, lanes 1–4). Specifically for
the HeLaEYFP-U2AF35cell line, the antibody additionally
recognizes an EYFP-tagged U2AF35 band of ?60 kDa
(Fig. 2A, lane 3). The monoclonal antibody MC3, directed
against U2AF65 (Gama-Carvalho et al. 1997), recognizes a
single band of ?53 kDa for endogenous U2AF65 (Fig. 2B,
lanes 1–4) and, specifically in the HeLaEYFP-U2AF65cell line,
an additional band of ?83 kDa for EYFP-U2AF65 (Fig. 2B,
lane 4). Probing with an anti-GFP-antibody results in a
single FP-positive band at ?30 kDa for EGFP in HeLaEGFP
(Fig. 2C, lane 2), a ?60 kDa band for EYFP-U2AF35 in
HeLaEYFP-U2AF35(Fig. 2C, lane 3), and a ?83 kDa signal for
EYFP-U2AF65 in HeLaEYFP-U2AF65(Fig. 2C, lane 4), respec-
tively. As expected, no bands in the parental HeLa cell
line are detected with anti-GFP (Fig. 2C, lane 1). HeLa
cells constitutively expressing EYFP-U2AF35 and EYFP-
U2AF65, respectively, express full-length FP-protein at a
similar or lower level to endogenous U2AF (Fig. 2A, lane 3;
Fig. 2B, lane 4).
To investigate whether the expression of the FP-fusion
proteins affected cell cycle progression, the growth proper-
ties of HeLaEYFP-U2AF35and HeLaEYFP-U2AF65cells were com-
pared with those of both parental HeLa and HeLaEGFPcells.
FIGURE 1. U2AF35 and U2AF65 colocalize in nuclear speckles with
snRNP proteins and non-snRNP splicing factors. (A–C) HeLa cells
transiently expressing EYFP-U2AF35 were counterstained with anti-
U2AF65. (overlay in C) EYFP-U2AF35 colocalizes with endogenous
U2AF65. (E,F,overlay in G) HeLaEYFP-U2AF35cells were fixed and
immunostained with anti-Y12, which recognizes the Sm family of
snRNP proteins. (I,J,overlay in K) HeLaEYFP-U2AF65cells were fixed
and immunostained with anti-SC-35, demonstrating colocalization
with the non-snRNP protein splicing factor. (E,I) Stable cell lines
HeLaEYFP-U2AF35and HeLaEYFP-U2AF65show identical localization
patterns as transiently expressing cells. (D,H,L) Cells were DAPI-
stained to visualize the cell nucleus. Bars, 10 mm.
FRET analyses of the U2AF complex
The growth rates of the respective cell lines are equivalent
and, as confirmed by FACS analyses, the relative populations
in the G1, S, and G2/M cell cycle stages are similar (Fig. 2D–
G). We conclude that expression of the FP-fusion proteins
in these cell lines does not prevent normal rates of either cell
division or cell cycle progression.
EYFP-U2AF35 and EYFP-U2AF65 bind
pre-mRNA in vitro
To confirm biochemically that the EYFP-fusion proteins of
U2AF35 and U2AF65 are functional, we investigated whether
the fusion proteins bind to pre-mRNA and whether nuclear
extracts prepared from stable cell lines expressing the fusion
proteins support pre-mRNA splicing in vitro. Nuclear ex-
tracts obtained from stable cell lines HeLaEYFP-U2AF35and
HeLaEYFP-U2AF65were analyzed in in vitro splicing assays
(Fig. 3, lanes 4,5), and their splicing activities were compared
with those of nuclear extracts from parental HeLa cells
and HeLaEGFPstable cells (Fig. 3, lanes 2,3). For both
HeLaEYFP-U2AF35and HeLaEYFP-U2AF65cell lines, we observed
equivalent pre-mRNA splicing efficiency as with control
extracts. The stably expressed fusion proteins thus do not
act as dominant-negative inhibitors of splicing, as may be
expected if the fusion proteins were misfolded or otherwise
inactive. To confirm that the FP-fusion proteins bind pre-
mRNA during spliceosome assembly, duplicates of the
splicing reactions were immunoprecipitated using anti-GFP
coupled Protein G–Sepharose. Following Proteinase K diges-
tion, any coimmunoprecipitated RNA species were resolved
on a denaturing RNA gel (Fig. 3, lanes 6–10). These data
show that both stably expressed EYFP-U2AF35 (Fig. 3, lane
9) and EYFP-U2AF65 (Fig. 3, lane 10) bind pre-mRNA in in
vitro splicing conditions but are not recovered with splicing
intermediates or products. This observation is consistent
with previous studies in which the U2AF subunits were
found specifically in complexes formed during early steps of
spliceosome assembly (Zamore et al. 1992; Merendino et al.
1999; Wu et al. 1999; Zorio and Blumenthal 1999). Stably
expressed EGFP alone does not bind pre-mRNA in splicing
tionof splicing reactions with beads without anti-GFP resulted
in no signals on the RNA gel (Fig. 3, lanes 6,7), ruling out
nonspecific binding of the pre-mRNA to the beads. Based
upon the above combination of biochemical assays and fluo-
rescence localization data, we conclude that the FRET analysis
of the respective FP-tagged fusion proteins is likely to reflect
interactions in vivo between the endogenous proteins.
FRET microscopy analyses show that U2AF35
and U2AF65 interact directly in vivo
In these experiments ECFP and EYFP serve as the donor–
acceptor pair for FRET. If donor (ECFP) and acceptor
FIGURE 2. Characterization of stable cell lines HeLaEYFP-U2AF35and HeLaEYFP-U2AF65. (A,B) HeLa cells expressing EYFP-U2AF35 and EYFP-
U2AF65, respectively, express full-length FP-protein at a similar level to endogenous U2AF. Nuclear extracts (25 mg total protein) from parental
HeLa cells (lane 1) and stable cell lines HeLaEGFP(lane 2), HeLaEYFP-U2AF35(lane 3), and HeLaEYFP-U2AF65(lane 4) were probed on Western blots
with anti-U2AF35 (A), anti-U2AF65 (B), and anti-GFP (C). FACS analyses of synchronized cell populations stained with propidium iodide show
similar distribution in cell cycle stages for parental HeLa (D) and stable cell lines HeLaEGFP(E), HeLaEYFP-U2AF35(F), and HeLaEYFP-U2AF65(G).
1204RNA, Vol. 11, No. 8
Chusainow et al.
(EYFP) are in close proximity (<10 nm) and in the appro-
priate relative orientation to each other, excitation of the
donor molecule leads to transfer of energy to the acceptor.
This energy transfer results in a decrease in emission from
the donor and an increase in fluorescence at the emission
wavelength of the acceptor. We measured FRET in two
different ways: (1) by imaging sensitized emission and (2)
by acceptor photobleaching. Both methods are based on
measuring fluorescence intensities. Detecting sensitized
emission is the technically more straightforward method
for measuring FRET, but it is the more complex one to
analyze. Although appropriate filter sets are used to isolate
the specific signals from donor and acceptor, the detected
FRET signal can be contaminated by spectral bleed-
through, which needs to be carefully corrected. Spectral
bleed-through is contributed by both the donor emission
that is detected in the acceptor (FRET) channel and by direct
excitation of the acceptor fluorophore at the wavelength used
to excite the donor. Therefore, donor-only and acceptor-only
controls were included in the measurements. Nonetheless,
variations in fluorophore concentration can complicate sensi-
tized emission measurements of FRET by affecting the level of
bleed-through, especially when studying transiently expressed
fusion proteins. The advantage of acceptor photobleaching
FRET microscopy is that only the donor fluorescence emission
is measured, comparing the quenched with the unquenched
donor emission after specific photobleaching of the acceptor
fluorophore. In this approach, problems associated with spec-
tral bleed-through and variations in fluorophore concentra-
tion are largely avoided.
1. Direct protein–protein interaction between U2AF35 and
U2AF65 detected by sensitized emission FRET microscopy
A strong FRET signal was observed in live cells transiently
coexpressing ECFP-U2AF65 and EYFP-U2AF35 (Fig. 4A–C),
proteins in vivo. Figure 4C shows the FRET signal corrected
for spectral bleed-through, FRETN, giving a diffuse nuclear
FIGURE 3. In vitro pre-mRNA splicing activity of stable cell lines
HeLaEYFP-U2AF35and HeLaEYFP-U2AF65, and binding of the FP-fusion
proteins to pre-mRNA in splicing conditions. Nuclear extracts obtained
from parental HeLa cells (lane 2) and stable cell lines HeLaEGFP(lane 3),
HeLaEYFP-U2AF35(lane 4), and HeLaEYFP-U2AF65(lane 5), respectively,
were tested in in vitro splicing assays as described in Materials and
Methods. Duplicates of the splicing reactions were subjected to immu-
noprecipitation using anti-GFP coupled Protein G–Sepharose. After
washing the beads, immunoprecipitated proteins were digested, and
coimmunoprecipitated RNA species were analyzed on a denaturing
RNA gel (lanes 6–10). Both stably expressed EYFP-U2AF35 (lane 9)
and EYFP-U2AF65 (lane 10) bind pre-mRNA in in vitro splicing con-
ditions but are not associated with splicing intermediates or products.
No RNA was coimmunoprecipitated with EGFP (lane 8), serving as
negative control. Incubation of splicing reactions with beads without
anti-GFP resulted in no signals on the RNA gel (lanes 6,7), ruling out
nonspecific binding of pre-mRNA to the beads. Lane 1 shows 50% of
the input pre-mRNA added to each splicing reaction.
FIGURE 4. In vivo detection of direct protein–protein interactions
between the U2AF subunits by sensitized emission FRET microscopy.
Live HeLa cells transiently coexpressing ECFP-U2AF65 and EYFP-
U2AF35 (A–C), ECFP-U2AF65 and EYFP-U2AF65 (D–F), or ECFP-
U2AF35 and EYFP-U2AF35 (G–I), respectively, were imaged on a
wide-field fluorescence microscope as described under Materials and
Methods. FRETN stands for spectral bleed-through corrected FRET
signal. ECFP-U2AF65 (A) and EYFP-U2AF35 (B) show a strong
FRET signal (C); ECFP-U2AF35 (G) and EYFP-U2AF35 (H) show
a slightly weaker but distinct FRET signal (I). No FRET (F) was
observed in cells coexpressing ECFP-U2AF65 (D) and EYFP-
U2AF65 (E), serving as negative control. All images are scaled to
0–4096 gray values (12-bit) and intensities colored as shown. FRETN
images are illustrated in pseudo-colors. Bars, 10 mm.
FRET analyses of the U2AF complex
pattern with the strongest intensities at nuclear speckles. The
same positive result was obtained when the fluorophores were
exchanged, i.e., so that ECFP-U2AF35 was the donor and
EYFP-U2AF65 the acceptor (data not shown). No detectable
FRET was measured in cells coexpressing ECFP-U2AF65 and
EYFP-U2AF65 (Fig. 4D–F), serving as a negative control.
As described below, we have detected a novel U2AF35
self-interaction in live cells coexpressing ECFP-U2AF35
and EYFP-U2AF35 (Fig. 4G–I).
2. Direct protein–protein interaction between U2AF35
and U2AF65 detected by acceptor photobleaching
Following photobleaching of the acceptor (EYFP-U2AF35) in
a transient enhancement in the donor
(ECFP-U2AF65) emission was observed
(Fig. 5). This dequenching effect indicates
an abolishment of FRET due to photo-
bleaching of the acceptor fluorophore,
and thus confirms that the two proteins
interact directly in vivo. Images were
acquired before (Fig. 5A–D) and after
(Fig. 5E,F) photobleaching the acceptor
(EYFP-U2AF35) with a single 150-msec
stationary laser pulse. The post-bleach
images, collected after 2 msec, clearly
show the dequenching effect of the
donor (Fig. 5E, arrow) as a result of
bleaching the acceptor (Fig. 5F, arrow).
acceptor into the bleach zone, de-
quenching of the donor was detectable
only for a short time period of ?1 sec.
No detectable dequenching was ob-
served in a nonbleached region (arrow-
head) similar in intensity and structure
to the bleached region. In Figure 5G,
donor and acceptor mean signal inten-
sities, monitored in the bleached and
nonbleached regions, respectively, were
plotted over time. Consistent with the
results obtained by sensitized emission
FRET microscopy, FRET between FP-
fusions of U2AF65 and U2AF35 re-
mained when the fluorophores were
switched. Figure 5H shows a diagram
obtained with the different protein pair
combinations. There was little or no
difference in FRET efficiencies detected
from speckled regions compared with
arguing that the interaction exists in
both the speckles and the nucleoplasm. For the interaction
between FP-U2AF35 and FP-U2AF65, FRET efficiencies of
16.4% and 21.0% were observed for the two combinations,
respectively. A FRET efficiency of 2.6%, observed for ECFP-
U2AF65 and EYFP-U2AF65, was considered as a negative
result. A FRET efficiency <5% is generally taken to be not
FRET microscopy reveals a novel self-interaction
of U2AF35 in vivo
Alongside FRET studies analyzing the direct interaction
between U2AF35 and U2AF65 in vivo, we have also detected
a novel U2AF35 self-interaction. A clear FRET signal was
observed in live cells transiently coexpressing ECFP-U2AF35
and EYFP-U2AF35, both by detecting sensitized emission
FIGURE 5. In vivo detection of direct protein–protein interactions between the U2AF sub-
units by acceptor photobleaching FRET microscopy. Live HeLa cells transiently coexpressing
ECFP-U2AF65 (A,C,E) and EYFP-U2AF35 (B,D,F) were analyzed on a wide-field fluorescence
microscope equipped with a quantifiable laser module as described under Materials and
Methods. Images were acquired before (A–D) and after (E,F) photobleaching the acceptor
(EYFP-U2AF35) with a single 150-msec stationary laser pulse. Only the first post-bleach
images of donor and acceptor collected after 2 msec are displayed here. A nonbleached region
(arrowhead) similar to the bleached region (arrow) was included in the data analysis for
comparison. (C–F) Images are illustrated in pseudo-colors. Bar, 10 mm. (G) Donor and
acceptor mean signal intensities monitored in the bleached and nonbleached regions were
plotted over time. Photobleaching the acceptor fluorophore abolished FRET resulting in an
enhancement in the donor emission because of dequenching. Because of a rapid recovery of
the bleached acceptor, dequenching of the donor was detectable for a short time period of
?1 sec only. (H) Acceptor photobleaching measurements were carried out for different protein
pair combinations of ECFP-U2AF35 (C35), EYFP-U2AF35 (Y35), ECFP-U2AF65 (C65), and
EYFP-U2AF65 (Y65), and the calculated FRET efficiencies are summarized in a diagram. For
the interaction between FP-U2AF35 and FP-U2AF65 FRET, efficiencies of 16.4% and 21.0%
were observed for the two combinations, respectively. A lower but distinct FRET efficiency of
8.2% was calculated for the U2AF35 self-interaction. A FRET efficiency of 2.6% observed for
ECFP-U2AF65 and EYFP-U2AF65 was considered to be not significant. n=7 cells for each
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(Fig. 4G–I) and by acceptor photobleaching FRET micros-
copy. For the U2AF35 self-interaction, the FRET efficiency
was calculated to be 8.2% (Fig. 5H). As observed for the
interaction between U2AF35 and U2AF65, there was no
apparent difference in FRET efficiencies detected from
speckled regions as compared with the values obtained
from diffuse nucleoplasmic regions.
Interaction between U2AF35 and U2AF65 as
well as the U2AF35 self-interaction persist in live
cells when transcriptional activity is inhibited
To investigate whether the protein–protein interaction
between the U2AF subunits U2AF35 and U2AF65 and the
U2AF35 self-interaction are dependent on splicing activity,
live HeLa cells coexpressing the respective FP fusion proteins
were treated with 5,6-dichloro-1-b-d-ribofuranosylbenzi-
midazole (DRB) to inhibit transcription,
and hence splicing activity, before ana-
yzing the protein–protein interactions by
acceptor photobleaching FRET micros-
DRB treatment of live HeLa cells co-
expressing ECFP-U2AF65 and EYFP-
U2AF35 resulted in a nuclear localization
pattern typical for splicing protein factors
in cells when transcription is inhibited;
i.e., the pattern shows enlarged nuclear
speckles, distinct from a dimmer, non-
speckled nucleoplasmic staining (Fig. 6).
As in transcriptionally active cells, photo-
bleaching the acceptor (EYFP-U2AF35)
led to a transient enhancement in donor
(ECFP-U2AF65) emission, confirming
that the two proteins interact directly
(Fig. 6). Images were acquired before
(Fig. 6A–D) and after (Fig. 6E,F) photo-
bleaching the acceptor (EYFP-U2AF35)
with a single 150-msec stationary laser
pulse. The post-bleach images, collected
after 2 msec, clearly show the dequench-
ing effect of the donor (Fig. 6E, arrow)
as a result of bleaching the acceptor (Fig.
6F, arrow). No detectable dequenching
was observed in a nonbleached region
(arrowhead) similar in intensity and
structure to the bleached region. In Fig-
ure 6G, donor and acceptor mean signal
intensities monitored in the bleached and
nonbleached regions were plotted over
time. FRET efficiencies obtained from
acceptor photobleaching the enlarged
speckles were compared with those
obtained from the diffuse nucleoplasmic
pool. The results are summarized in a
diagram in Figure 6H. In cells coexpressing ECFP-U2AF65
and EYFP-U2AF35 where transcriptional activity was inhib-
ited, positive FRET values were observed in both the enlarged
speckles and the diffuse nucleoplasmic pool. The same obser-
copy was carried out with cells coexpressing ECFP and EYFP
fusion proteins of U2AF35 and inhibited in transcriptional
activity (results summarized in Fig. 6H). In contrast to cells
coexpressing ECFP-U2AF65 and EYFP-U2AF35, where FRET
lar values, as statistically confirmed by applying the Student’s
t-test to the data (mean values not significantly different with
U2AF35, the FRET efficiencies measured at speckles were
significantly higher than those measured in the nucleoplas-
mic pool (means significantly different with P=0.005,
n=3). In general, FRET efficiencies obtained from cells
FIGURE 6. In vivo detection of direct protein–protein interactions between the U2AF sub-
units in live cells after blocking transcription. Live HeLa cells transiently coexpressing ECFP-
U2AF65 (A,C,E) and EYFP-U2AF35 (B,D,F) were treated with DRB as described in Material
and Methods in order to block transcription and analyzed by acceptor photobleaching FRET
microscopy. Images were acquired before (A–D) and after (E,F) photobleaching the acceptor
(EYFP-U2AF35) with a single 150-msec stationary laser pulse. (E,F) The first post-bleach
images of donor and acceptor collected after 2 msec. A nonbleached region (arrowhead)
similar to the bleached region (arrow) was included in the data analysis for comparison.
(C–F) Images are illustrated in pseudo-colors. Bar, 10 mm. (G) Donor and acceptor mean
signal intensities monitored in the bleached and nonbleached regions were plotted over time.
Photobleaching the acceptor fluorophore abolished FRET, resulting in an enhancement in the
donor emission because of dequenching. Because of a rapid recovery of the bleached acceptor,
dequenching of the donor was detectable for a short time period of ?1 sec only. Measurements
were focused on nuclear speckles and diffused nucleoplasmic regions for comparison. (H)
FRET efficiencies were calculated and summarized in a diagram. Results, obtained from
measurements with DRB-treated cells coexpressing ECFP-U2AF35 and EYFP-U2AF35, are
also summarized in H. For the interaction between ECFP-U2AF65 and EYFP-U2AF35 in
DRB-treated cells, FRET efficiencies of 29.7% in nuclear speckles and 26.3% in the diffused
nucleoplasmic pool were observed. For the U2AF35 self-interaction in DRB-treated cells FRET
efficiencies of 19.7% in nuclear speckles and 11.8% in the diffused nucleoplasmic pool were
observed. n=7 cells for each measurement.
FRET analyses of the U2AF complex
inhibited in transcriptional activity had higher values than
those from nontreated cells. There are several possible rea-
sons for the observed variations in FRET efficiencies. A
possible explanation is that it results from the change in
local concentration of FP-U2AF in speckles, which occurs
when transcription is inhibited. The fluorophore concentra-
tion is a critical factor in intensity-based imaging as changes
in donor and acceptor concentrations will affect the FRET
measurement, in particular if the concentrations of the two
fluorophores change to different extents. Furthermore, if the
intensities are very low, an unfavorable signal-to-noise ratio
can lead to an alteration in the FRET efficiency that is
In summary, these data show that both the interaction
between U2AF35 and U2AF65 and the U2AF35 self-
interaction exist independent of transcription levels in vivo
and hence must occur in the presence of different levels of
splicing activity. The observation that positive FRET values
were obtained in both the enlarged speckles and the diffuse
nucleoplasmic pool upon inhibition of transcription is con-
sistent with U2AF complexes being preassembled before pre-
mRNA binding and recruitment into the spliceosome. Taken
together, the above findings support the idea that nuclear
speckles may play a role in storage and preassembly of splice-
osome components (Spector 1993; Zhang et al. 1994).
Biochemical assays confirm a U2AF35
self-interaction in vitro
next carried out biochemical assays to test whether this inter-
action could also be detected in vitro (Fig. 7). In a coimmuno-
precipitation assay (Fig. 7A), nuclear extracts from parental
HeLa cells, and HeLaEGFPand HeLaEYFP-U2AF35stable cell
lines, respectively, were incubated with Protein G–Sepharose
coupled to anti-GFP antibodies. After washing the beads,
immunoprecipitated proteins were separated by SDS-PAGE,
blotted onto a nitrocellulose membrane, and probed with
an anti-peptide antibody specific for U2AF35. Endogenous
U2AF35 was coimmunoprecipitated from HeLaEYFP-U2AF35
nuclear extracts (Fig. 7A, lane 5), resulting in a distinct protein
band at ?30 kDa, in addition to the fusion protein signal at
?60 kDa. No endogenous U2AF35 was immunoprecipitated
from parental HeLa (Fig. 7A, lane 3) or HeLaEGFP(Fig. 7A,
lane 4) cells, respectively, serving as negative controls. Further-
was incubated with beads without anti-GFP antibodies, result-
of U2AF35 to the beads.
In a GST pull-down assay (Fig. 7B), in vitro translated,
35S-radiolabeled U2AF35 was incubated with either recombi-
nant protein GST-U2AF35 or GST-U2AF65, respectively,
and captured on glutathione–Sepharose beads and washed,
and the bound fractions were analyzed by SDS-PAGE and
detected by autoradiography. In vitro translated U2AF35 was
copurified with GST-U2AF35 (Fig. 7B, lane 3) and GST-
U2AF65 (Fig. 7B, lane 4), but not with GST alone (Fig. 7B,
lane 2), serving as a negative control. A separate negative
FIGURE 7. U2AF35 self-interaction analyzed in vitro. (A) Endogen-
HeLaEYFP-U2AF35cells. FP-tagged proteins were immunoprecipitated
from nuclear extracts using anti-GFP antibodies, and the immuno-
precipitates were probed on a Western blot for U2AF35. Endogenous
U2AF35 was coimmunoprecipitated from HeLaEYFP-U2AF35nuclear
extracts (lane 5) but not from parental HeLa (lane 3) or HeLaEGFP
(lane 4) cells, respectively, serving as negative controls. A control
reaction, where HeLaEYFP-U2AF35nuclear extract was incubated with
beads without anti-GFP, resulted in no signals (lane 2). Ten percent
immunoprecipitation input amount of HeLaEYFP-U2AF35nuclear ex-
tract was separated in lane 1 for comparison. (B) In vitro translated
U2AF35 copurifies with recombinant GST-U2AF35 protein. In vitro
translated,35S-radiolabeled U2AF35 was incubated with recombinant
protein GST-U2AF35 (lane 3) or GST-U2AF65 (lane 4), respectively,
and captured on glutathione–Sepharose beads, and the bound frac-
tions were analyzed by SDS-PAGE and detected by autoradiography.
In vitro translated U2AF35 was co-purified with GST-U2AF35 (lane
3) and GST-U2AF65 (lane 4) but not with GST (lane 2), serving as a
negative control. A control reaction, where in vitro translated U2AF35
was incubated with beads only, resulted in no signal (lane 1). (C)
Recombinant His-tagged U2AF35 interacts with recombinant GST-
U2AF35 protein. 6?His-U2AF35 was incubated with GST-U2AF35
(lane 3) or GST-U2AF65 (lane 4), respectively, captured on glu-
tathione–Sepharose beads, and the bound fractions were probed on
a Western blot for His-tagged protein. 6?His-U2AF35 was co-purified
with GST-U2AF35 (lane 3) and GST-U2AF65 (lane 4) but not with
GST (lane 2), serving as negative control. A control reaction, where
His-tagged U2AF35 was incubated with beads only, resulted in no
signals (lane 1).
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Chusainow et al.
control reaction, where in vitro translated U2AF35 was incu-
bated with beads only, resulted in no signals (lane 1), ruling
out nonspecific binding of the in vitro translated protein to
Consistent results were obtained in a similar GST pull-
down assay where recombinant His-tagged U2AF35 was
incubated with either GST-U2AF35 or GST-U2AF65, respec-
tively, and captured on glutathione–Sepharose beads and
washed, and the bound fractions were analyzed by SDS-
PAGE, blotted onto a nitrocellulose membrane, and probed
withS-protein HRP conjugate. 6?His-U2AF35 was copurified
with GST-U2AF35 (Fig. 7C, lane 3) and GST-U2AF65 (Fig.
7C, lane 4), but not with GST alone (Fig. 7C, lane 2), serving
as a negative control. A separate negative control reaction,
where His-tagged U2AF35 was incubated with beads only,
resulted in no signals (Fig. 7C, lane 1). In summary, the
biochemical assays confirm the existence of a U2AF35 self-
interaction in vitro, consistent with the in vivo FRET data.
While the cellular function of the U2AF35 self-interaction
remains to be determined, the fact that it can be detected in
vivo in live cells, as well as in vitro, indicates that it may be
physiologically significant. The existence of a U2AF35 self-
interaction suggests that the stoichiometry of the U2AF
complex may, at least in part, differ in vivo from the pre-
viously described heterodimeric complex. For example, both
homodimeric and heterodimeric forms of U2AF35 may
coexist and higher-order complexes could also form in
vivo. In this regard it is notable that the RS domain of
U2AF35, but not U2AF65, interacts with other splicing
factors (Wu and Maniatis 1993). It will be interesting in
the future to test whether other SR proteins can also form
self-interactions similar to U2AF35.
Our data contrast with the results obtained in a previous
study where no self-interaction of U2AF35 was observed in
protein binding assays (Tronchere et al. 1997). This apparent
discrepancy could be due to the different conditions used in
the respective binding assays. For example, we observed that
the U2AF35 self-interaction appears weaker than the
U2AF65–U2AF35 interaction. Therefore, the self-interaction
may not be detected if more stringent assay conditions are
used, which nonetheless retain the U2AF65–U2AF35 inter-
action. The fact that this study has revealed a novel self-
interaction within the extensively studied U2AF complex
emphasizes the potential that FRET microscopy techniques
have to uncover new protein–protein interactions that may
be overlooked by conventional methods.
MATERIALS AND METHODS
FP-U2AF35 constructs were generated by EcoR1/BamH1 diges-
tion and gel extraction from pEGFP-U2AF35 and ligation into
pECFP-C1 and pEYFP-C1 (Clontech). pEGFP-U2AF35 was ob-
tained by PCR amplification from pGEM-U2AF35 (gift from
Phillip D. Zamore, Univ. of Massachusetts Medical School) using
the primers 50-GCGGATCCTGGCTCAGAATCGCCCAGATCT-30
(forward) and 50-GCGAATTCAATGGCGGAGTATCTGGCCTCC-
30(reverse) and subcloned into pEG FP-C1 (Clontech) digested with
EcoR1/BamH1. FP-U2AF65 constructs were gained by PCR amplifi-
cation from pHb-U2AF65 (gift from Juan Valcarel, CRG, Barcelona,
Spain) using the primers 50-GCGGATCCTCTACCAGAAGTCCCG
GCGGTG-30(forward) and 50-CGGAATTCCATGTCGGACTTC
GACGAGTTC-30(reverse) and ligated into pECFP-C1 and pEYFP-
C1 digested with EcoR1/BamH1. The constructs pGEX-U2AF35 and
pET-U2AF35 were generated by PCR amplification from pEYFP-
U2AF35 using the primers 50-GCGGATCCATGGCGGAGTATCTG
GCCTC-30(forward) and 50-GAGAATTCGTCAGAATCGCCCAGA
TCTTTC-30(reverse) and ligated into pGEX-6P-3 (Amersham Bio-
sciences) and pET-30a(+) (Novagen) digested with BamH1/EcoR1.
pGEX-U2AF65 was gained by PCR amplification from pEYFP-
U2AF65 using the primers 50-CTGGATCCATGTCGGACTTCGAC
GAGTTC-30(forward) and 50-GAGAATTCTCTACCAGAAGTCC
CGGCGGTG-30(reverse) and subcloned into pGEX-6P-3 digested
with BamH1/EcoR1. All generated plasmid constructs described
above were confirmed by restriction analysis and sequencing.
Peptide antibody production
An anti-peptide antibody specific for U2AF35 was raised in rabbit
using the peptide sequence YRNPQNSSQSADGL and was affin-
ity-purified (Eurogentec). This sequence is located between the
zinc finger region and the RRM domain at the N terminus of the
protein and is a region that is unique to U2AF35 with little or no
similarity to other proteins. The affinity-purified peptide-anti-
body was tested on HeLa nuclear extracts by immunobloting
and on fixed HeLa cells by immunofluorescence microscopy.
Cell culture, transfection assay, and generation
of stable cell lines
HeLa cells were cultured as monolayers in Dulbecco’s modified
Eagle medium supplemented with 10% fetal bovine serum and
100 U/mL penicillin-streptomycin (Invitrogen Life Technologies).
For the expression and microscopic detection of fluorescent
fusion proteins in vivo, cells were grown on glass coverslips and
transfected with 1 mg of the appropriate plasmid DNA using
Effectene tranfection reagent (QIAGEN) according to the manu-
facturer’s instructions. Cell fixation or live cell microscopy was
carried out at 8–12 h post-transfection. Stable cell lines expressing
EYFP-U2AF35 and EYFP-U2AF65, respectively, were generated
using G418 selection of transiently transfected HeLa cells essen-
tially as described previously (Sleeman et al. 2001).
Cell fixation, immunofluorescence microscopy,
and FACS analyses
HeLa cells grown on glass coverslips were fixed for 5 min in freshly
prepared 3.7% paraformaldehyde in 37?C PHEM buffer (60 mM
PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2[pH 6.9]).
Permeabilizing was performed with 1% Triton X-100 in phosphate
buffered saline (PBS) for 10 min at room temperature. After block-
ing the cells with 1% serum (goat) in PBS with 0.05% Tween 20
FRET analyses of the U2AF complex
(BDH) for 20 min, they were incubated with primary antibody
diluted in PBS/Tween 20 with 1% serum for 1 h, washed, and
incubated with the appropriate diluted secondary antibody for
45 min. If required, cells were stained with DAPI (0.3 mg/mL,
Sigma). After a final set of washes with PBS, cells were mounted in
Vectashield medium (Vector Laboratories). Antibodies used were
mouse mAb MC3, which is specific for U2AF65 (dilution 1:10)
(Gama-Carvalho et al. 1997), mouse Y12 mAb anti-Sm (dilution
1:500) (Lerner et al. 1981; Pettersson et al. 1984), and mouse mAb
directed against SC-35 (dilution 1:2000, Sigma-Aldrich) (Fu and
Maniatis 1990). TRITC-conjugated goat anti-mouse IgG (dilution
1:500, Jackson ImmunoResearch Laboratories) was used as second-
Three-dimensional images of fixed immunostained cells were
recorded on a Zeiss Axiovert-DeltaVision Image Restoration
Microscope (Applied Precision) as previously described (Platani
et al. 2000) using a 1003 numerical aperture (NA) 1.4 Plan-
Apochromat objective and running SoftWorx (Applied Precision)
imaging and deconvolution software.
For FACS analyses parental HeLa cells, HeLaEGFP(Trinkle-
Mulcahy et al. 2003), HeLaEYFP-U2AF35, and HeLaEYFP-U2AF65
cells were harvested by trypsinization, washed with PBS, and
fixed in 70% ethanol for 3 h at 4?C. After staining the cells
with 25 mg/mL propidium iodide in PBS containing 100 mg/mL
RNase A, fluorescence was measured using a FACSort (Becton
Dickinson), and data were analyzed using Cell Quest software
FRET measurements were implemented in two different ways: (1)
by imaging sensitized emission and (2) by acceptor photobleach-
ing. All measurements were carried out on live cells.
Sensitized emission FRET microscopy was performed basically
as described previously (Trinkle-Mulcahy et al. 2001). HeLa cells
grown on glass coverslips were cotransfected with an ECFP con-
struct of U2AF35 or U2AF65, respectively, and an EYFP construct
of U2AF35 or U2AF65 (Table 1). Eight to 12 h post-transfection,
cells were mounted in HEPES buffered Phenol red–free medium
(Invitrogen Life Technologies) in a closed,
heated chamber (Bachofer) and imaged on
the DeltaVision system (described above)
using specific excitation and emission filters
(Chroma) to resolve the ECFP and EYFP
signals and running SoftWorx imaging soft-
ware. To correct for spectral bleed-through
and for uncontrolled variations in donor–
acceptor concentrations, a combination of
donor, FRET, and acceptor filter sets was
used to isolate and maximize three specific
signals: donor fluorescence, acceptor fluo-
rescence resulting from FRET, and the
directly excited acceptor fluorescence, re-
spectively. Filter sets used were as follows:
excitation 436/10 nm and emission 470/30
nm for ECFP, excitation 500/20 nm and
emission 535/30 nm for EYFP, and excita-
tion 436/10 nm and emission 535/30 nm for
FRET, and a dichroic filter JP4 (multiband
beam splitter for ECFP and EYFP). FRET was measured by excit-
ing cells at the ECFP (donor: excitation 433 nm, emission 475
nm) wavelength and detecting at the EYFP (acceptor: excitation
513 nm, emission 527 nm) emission wavelength. Three different
specimen, containing just donor, just acceptor, and both donor
and acceptor were examined with each of the three filter sets, and
the resulting data were corrected for spectral bleed-through by
using the following equation:
Net Energy Transfer
FRETN ¼ FRET signal ? aðdonor signalÞ ? bðacceptor signalÞ
For this equation, a and b were determined by imaging the cells
expressing each fusion protein on its own. In our system, ?46%
(SD 2%) of the cyan signal and 24% (SD 1%) of the yellow signal
was detected with the FRET filter set. These numbers represent
average values, each obtained from imaging 25 different cells.
To detect FRET by the method of acceptor photobleaching,
cells were prepared the same way as described above for
sensitized emission. Measurements were conducted on a Del-
taVision Spectris Image Restoration Microscope (Applied Pre-
cision) fitted with a Quantifiable Laser Module (QLM)
including a 20-mW 532-nm CW laser, suitable for photo-
bleaching YFP without cobleaching CFP. Images were collected
using an Olympus 603 NA 1.4 Plan-Apochromat lens, a Photo-
metrics CoolSnap HQ cooled CCD camera and SoftWorx
(Applied Precision) imaging software. The following specific
CFP/YFP filter sets were used to resolve the ECFP and EYFP
signals: excitation 436/10 nm and emission 480/40 nm for
ECFP; excitation 525/20 nm and emission 580/70 nm for
EYFP. The dichroics used were custom-built by Applied Pre-
cision and Chroma Technology. The set is modified from the
normal CFP/YFP JP4 set such that the dichroic reflects and
the emission filter rejects light at 532 nm, allowing this wave-
length to be used for selectively photobleaching YFP. After
obtaining three pre-bleach images, a defined region of the
cell nucleus was spot photobleached with a single 150-msec
stationary pulse at 90% laser power. The first image was
acquired 1–2 msec after the bleach event. For the first second,
images were acquired approximately every 200 msec; for the
TABLE 1. Plasmid constructs used and generated in this study
Starting material for cloning FP-U2AF35 constructs
Starting material for cloning FP-U2AF65 constructs
Expression of cyan fluorescent fusion proteins in
mammalian cells for fluorescence imaging,
including FRET studies
Expression of yellow fluorescent fusion proteins
in mammalian cells for fluorescence imaging
and generation of stable cell lines
Expression of recombinant GST-tagged
proteins in bacteria
Expression of recombinant 6?His-tagged
protein in bacteria; in vitro
1210 RNA, Vol. 11, No. 8
Chusainow et al.
following 1.7 sec, every 335 msec; and then at 830-msec intervals
in the following 5 sec, after which images were acquired every 1.6
sec for the remainder of the experiment. A total of 20 images
were acquired after the bleach event. Images of donor (ECFP)
and acceptor (EYFP) were taken in separate subsequent measure-
ments, bleaching exactly the same spot before collecting post-
bleach images. Obtained data were analyzed using the image
analysis tools included in the SoftWorx software and the biosta-
tistics program GraphPad Prism. In addition to the bleached
region, a similar nonbleached nuclear region in the same cell
was included in the data analysis as a control. A region of back-
ground fluorescence was defined outside the cell and subtracted
from both the bleached and control regions. The data were
normalized against the mean intensity of the whole image over
time to account for any fluctuations and normal photobleaching
that occur during image acquisition throughout the course of the
experiment. FRET efficiency was calculated by the following for-
E ¼ ðIDðpostÞ? IDðpreÞÞ=IDðpostÞ
where ID(pre) and ID(post) are donor intensity before and after
For inhibition of transcriptional activity, cells were treated for
2 h with 25 mg/mL DRB before carrying out FRET analyses by
Preparation of nuclear extracts, immunoblotting,
Nuclear extracts were prepared from parental HeLa cells and stable
cell lines as described previously (Ajuh et al. 2002). Twenty-five
micrograms of nuclear protein was electrophoresed on a Novex
12% tris-glycine polyacrylamide gel using MOPS running buffer
(Invitrogen) and transferred to Hybond-C Extra nitrocellulose
membrane (Amersham Biosciences) for immunoblotting. Follow-
ing blocking with 5% milk powder in PBS-Tween (0.3% Tween 20
in PBS), the membranes were incubated with primary antibodies
diluted in PBS-Tween containing 5% milk powder for 1 h. Primary
antibodies used were rabbit peptide antibody anti-U2AF35
(described above; dilutuion 1:10,000 in PBS), mouse mAb MC3
directed against U2AF65 (dilution 1:500) (Gama-Carvalho et al.
1997), and mouse mAb anti-GFP (dilution 1:1000, Roche). After
washing the membranes in PBS-Tween containing 5% milk pow-
der, they were incubated with the appropriate diluted secondary
antibody for 45 min. Secondary antibodies used were horseradish
peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-con-
jugated goat anti-mouse IgG (dilution 1:5000, Pierce). Detection
was performed via chemiluminescence by using ECL reagents
Nuclear extracts from parental HeLa cells, HeLaEGFP(Trinkle-
Mulcahy et al. 2003) and HeLaEYFP-U2AF35cells were precleared by
incubation with 10 mL Protein G–Sepharose (Amersham Bio-
sciences) for 1 h on a shaking platform. Fifty microliters of
precleared extract was incubated with 50 mL Protein G–Sepharose
coupled to 10 mg anti-GFP mouse monoclonal antibody (Roche)
in 400 mL binding buffer (20 mM HEPES [pH 7.9], 0.1 M KCl, 0.2
mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) for 4 h, rotating end
over end at 4?C. The beads were then washed four times with 250
mM NaCl, 20 mM Tris-HCl (pH 7.5), and 0.1% Triton X-100.
The immunoprecipitated proteins and an equal amount of cleared
input nuclear extract were separated on a 12% tris-glycine poly-
acrylamide gel, transferred to nitrocellulose membrane, and immu-
In vitro transcription/translation, expression,
and purification of recombinant proteins and
GST pull-down assays
35S-labeled U2AF35 protein was generated from pET-U2AF35
plasmid DNA by in vitro transcription/translation using a TNT
T7 rabbit reticulocyte lysate system (Promega) following the
Recombinant proteins GST-U2AF35 and GST-U2AF65 were
expressed from pGEX-U2AF35 and pGEX-U2AF65, respectively,
in Escherichia coli BL21 (DE3) (Novagen) and purified on glu-
tathione–Sepharose (Amersham Biosciences) essentially as de-
scribed previously (Ajuh et al. 2001) except that post induction
with 0.04 mM isopropyl-1-thio-bD-galactopyranoside (IPTG),
bacteria cultures were grown at room temperature for 1 h before
lysing them. 6?His-U2AF35 recombinant protein was expressed
from pET-U2AF35 in E. coli BL21 (DE3) pLysS (Novagen) and
purified on nickel-nitrilotriacetic acid-agarose (QIAGEN) essen-
tially as described previously (Ajuh et al. 2001) except that post-
induction with 1 mM IPTG, cells were grown at room temperature
for 2 h prior lysis.
incubated with equimolar amount of 6?His-U2AF35 recombinant
protein or 3 mL of35S-labeled U2AF35 in vitro translated protein,
respectively, in 7 mL binding buffer (350 mM NaCl, 20 mM Tris-HCl
[pH 7.5], 0.1% Triton X-100, 1 mg/mL bovine serum albumin) and
4?C on a shaking platform. The beads were washed four times with
350 mM NaCl, 20 mM Tris-HCl (pH 7.5), and 0.1% Triton X-100.
Bound proteins were separated on a 12% tris-glycine polyacryl-
amide gel. 6?His-U2AF35 protein was transferred to nitrocellulose
membrane and detected by using S-protein HRP conjugate (Nova-
gen) following the manufacturer’s instructions. To detect
labeled U2AF35, gel was fixed in 50% methanol and 10% acetic
acid for 30 min. Fixed gel was soaked in Amplify fluorographic
er for 30 min and then dried. Detection was performed by auto-
In vitro pre-mRNA splicing assay and
immunoprecipitation of the splicing reaction
Splicing assays were performed using uniformly labeled, capped
pre-mRNAs incubated with nuclear extracts using the in vitro
splicing conditions described previously (Lamond et al. 1987).
Adenovirus major late precursor (adeno pre-mRNA) was
transcribed from Sau3A-digested plasmid pBSAd1 (Konarska
and Sharp 1987). Splicing products were separated on a 10%
polyacrylamide/8 M urea denaturing gel using Tris-borate/EDTA
electrophoresis buffer and detected by autoradiography.
FRET analyses of the U2AF complex
For each immunoprecipitation reaction, 50 mL Protein G–
Sepharose coupled to 10 mg anti-GFP monoclonal antibody
(Roche) was preblocked to minimize nonspecific binding. This
was done by incubating the beads in 1 mL blocking buffer (20 mM
HEPES [pH 7.9], 0.3 M KCl, 0.01% Nonidet P40, 50 mg/mL
glycogen, 50 mg/mL tRNA, 1 mg/mL BSA) for 30 min rotating
at 4?C. Subsequently, the beads were washed four times with
binding buffer (20 mM HEPES [pH 7.9], 0.15 M KCl, 0.1%
Nonidet P40, 1 mg/mL BSA). Splicing reactions prepared with
nuclear extracts from parental HeLaEGFP(Trinkle-Mulcahy et al.
2003), HeLaEYFP-U2AF35, and HeLaEYFP-U2AF65cells, respectively,
were incubated with preblocked beads in 400 mL binding buffer
for 3 h rotating at 4?C, followed by four washes with 150 mM
NaCl and 20 mM Tris-HCl (pH 7.5). In order to elute and digest
immunoprecipitated protein and extract coimmunoprecipitated
RNA, the beads were incubated in 90 mL elution buffer (2% SDS,
20 mM Tris-HCl [pH 7.5], 20 mM DTT), containing 300 mg
Proteinase K and 12 mg glycogen, for 45 min at 65?C. The RNA
was ethanol-precipitated, resolved on a denaturing RNA gel as
described above, and visualized by autoradiography.
We thank Phillip D. Zamore for the construct pGEM-U2AF35 and
Juan Valcarcel for the pHb-U2AF65 plasmid. We are also grateful
to Jason R. Swedlow for technical help with the acceptor photo-
bleaching measurements. Thanks to all members of the Lamond
laboratory and the Ellenberg group for support. A.I.L. is a Well-
come Trust Principal Research Fellow. J.C. was supported by a
Biotechnology and Biological Sciences Research Council fellowship.
Received December 20, 2004; accepted April 29, 2005.
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