Nucleic Acids Research, Vol. 20, No. 14
3625-3630
U1 - U 2 snRNPs interaction induced by an RNA
complementary to the 5' end sequence of U1 snRNA
Marie-Claire Daugeron, Jamal Tazi, Philippe Jeanteur, Claude Brunei* and Guy Cathala
UA CNRS 1191, Genetique Moleculaire, Laboratoire de Biochimie, CRLC Val d'Aurelle-Paul
Lamarque, Pare Euromedecine, 34094 Montpellier Cedex 2 and Laboratoire de Biologie Moleculaire,
Universite Montpellier II, Sciences et Techniques du Languedoc, Place E. Bataillon, CP 012, 34095
Montpellier Cedex 05, France
Received April 8, 1992; Revised and Accepted June 25, 1992
ABSTRACT
Several lines of evidences indicate that U1 and U2
snRNPs become interacting during pre-mRNA splicing.
Here we present data showing that an U1 - U2 snRNPs
interaction can be mediated by an RNA only containing
the consensus 5' splice site of all of the sequences
characteristic of pre-mRNAs. Using monospecific
antibodies (anti-(U1) RNP and anti-(U2) RNP), we have
found that a tripartite complex comprising U1 and U2
snRNPs is immunoprecipitated In the presence of a
consensus 5' splice site containing RNA, either from
a crude extract or from an artificial mixture enriched
in U1 and U2 snRNPs. This complex does not appear
in the presence of an RNA lacking the sequence
complementary to the 5' terminus of U1 snRNA.
Moreover, RNAse T1 protection coupled to immunoprecipltation experiments have demonstrated that only
the 5' end sequence of U1 snRNA contacts the
consensus 5' splice site containing RNA, arguing that
U2 snRNP binding in the tripartite complex is mediated
by U1 snRNP.
INTRODUCTION
Splicing of pre-mRNAs occurs in a ribonucleoprotein structure
called the spliceosome. In vitro studies in both mammalian and
yeast systems based on affinity selection and non-denaturing gel
analyses have demonstrated that the abundant Ul, U2, U5 and
U4 —U6 ribonucleoprotein particles (U snRNPs) are among the
main components of the spliceosome apparatus (for recent
reviews, see 1 - 5 ) . The other actors are auxiliary non-snRNPs
proteins, whose exact number is not yet definitively established
and, almost certainly, ATP requiring enzymes whose association
with the spliceosome could be transient (6, 7).
A stepwise pathway of snRNPs and proteins binding to premRNAs is involved to assemble the functional spliceosome.
Concerning the snRNPs, current models hold that Ul and U2
snRNPs interact with the 5' splice site and the branchpoint
sequence, respectively, to give a pre-splicing complex and then
the spliceosome is formed upon addition of a pre-assembled multisnRNP complex comprising U4-U6 and U5 snRNPs (8-10).
As to the non-snRNP proteins, it is now well established that
some of them are involved at very early stages of spliceosome
assembly, for example U2AF (11, 12), pPTB (13), both being
most likely required for U2 snRNP binding, and SF2 (14, 15)
also called ASF for alternative splicing factor (16). Other proteins
have been described but their involvement in splicing is not as
well documented. These include the Al and C proteins (17 — 19)
as well as the so-designated intron binding proteins (20, 21),
which all interact specifically with the polypyrimidine tract/3'
splice site of pre-mRNAs, and some other components whose
involvement in splicing was evidenced by monoclonal antibodies
either raised against purified spliceosomes (22) or native hnRNP
(23).
However, the assembly of a functional spliceosome is most
likely much more complicated than previously thought. For
example, recent studies in the yeast Saccharomyces cerevisiae
system have revealed a role for the highly conserved UACUAAC
branchpoint sequence in the formation of commitment complexes
containing Ul snRNP (24-26). In mammals as well, targeted
depletion of Ul snRNP with 2'-0Me antisense RNA showed that
Ul snRNP most likely stabilizes U2 snRNP binding to the
branchpoint sequence in a fashion independent of the Ul
snRNA-5' splice site interaction (27).
Taking now into account these previously unrevealed early
Ul—U2 snRNPs interactions at the branchpoint sequence, we
asked whether, similarly, Ul and U2 snRNPs can together contact
the 5' splice site of pre-mRNAs. To this aim, we have used a
small synthetic RNA containing a sequence complementary to
the 5' terminus of Ul snRNA as a 5' splice site and have based
our investigation on the use of anti-(Ul) and anti-(U2) RNP
antibodies. Here we demonstrate that a fraction of Ul and U2
snRNPs interact together in the presence of such an RNA.
* To whom correspondence should be addressed at: UA CNRS 1191, Geneuque Moleculaire, University Montpellier U, Sciences et Techniques du
Languedoc, Place E. Bataillon, CP 012, 34095 Montpellier Cedex 05, France
3626 Nucleic Acids Research, Vol. 20, No. 14
MATERIALS AND METHODS
Materials
T7 RNA polymerase, RNasin, restriction enzymes and DNase
I (RNase-free) were from Promega Biotec, RNase Tl from
Calbiochem, ribonucleotides triphosphates from Boehringer
Mannheim, m 7 G5'pppG5' Cap from Pharmacia LKB
Biotechnology Inc., [a-nP] UTP and 32P orthophosphate from
Amersham Corp. All other chemicals were of analytical grade.
Plasmids, T7 transcription, nuclear extracts and oligodeoxynucleotide-directed cleavage of snRNAs
The ABPA3'T7 plasmid was constructed by inserting the HindlllEcoRI fragment from the ABPA3' plasmid previously described
(28) in pSP73 vector. Corresponding RNA was synthesized after
cutting the plasmid at the Bgin site. Conditions for transcription
were as previously described (20) using T7 RNA polymerase.
Preparation of HeLa cell nuclear extracts was as originally
described (29) in Triethanolamine buffer (20 mM TEA (pH 7.9),
20% [v/v] glycerol, 0.1 M KC1, 0.2 mM EDTA, 0.5 mM DTT)
(20). 32P-labeled nuclear extracts were prepared from cells
which were resuspended, to a concentration of 3 X lOVml, in 3 1
of phosphate-free MEM (eagle) medium supplemented with 5 %
non-dialyzed newborn calf serum and exposed to 10 mCi carrierfree H332PO4 (Amersham) for at least 15 h. Oligodeoxynucleotide cleavage of Ul and U2 snRNAs (nt 1 — 15) was as
described (28).
Isolation of Ul and U2 snRNPs by glycerol gradient
centrifugation
200 /tl of in vivo 32P labeled nuclear extract were two-fold
diluted with TEA buffer without glycerol and then layered onto
a 12 ml linear 10-30% [v/v] glycerol gradient in TEA buffer.
Centrifugation was in a Beckman SW 40 rotor at 29,000 rpm
for 18 hours (4°C). 24 fractions of 500 /tl were harvested from
top to bottom and analyzed for snRNA contents. Those enriched
in Ul and U2 snRNPs, respectively, were pooled, dialysed during
3 hours against TEA buffer containing 1 mM MgCb and finally
concentrated to about 200 /tl in Centricon 10 microconcentrators
(Amicon). Each concentrated pool was referred as 'purified' Ul
and 'purified' U2 snRNPs.
Immunoblots, immunoprecipitations and immunoselection of
RNase Tl RNA fragments
The patient anti-(U2) RNP (Ya), the mouse monoclonal anti-(U2)
RNP (4G3), the mouse monoclonal anti-(Ul) RNP (2.73), the
patient anti-(Ul,U2) RNP (pick U1-U2) and the anti-2,2,7
trimethyl guanosine antibodies were generous gifts respectively
from Dr. Mimori (Keio University, Japan), Dr. van Venrooij
(Nijmegen University), Dr. Hoch (Agouron institute, La Jolla),
Dr. Mattaj (EMBL, Heidelberg) and Dr. Luhrmann (IMT,
Marburg). The anti-(Ul) RNP serum Go was from the Centre
de Tranfusion Sanguine, Montpellier (Dr. H.Graafland). Proteins
from nuclear extracts were fractionated in SDS/10%
polyacrylamide gels and electrophoretically transferred to
nitrocellulose sheets (BA83 Schleicher & Schull) as described
(30). After blocking the sheet in TTBS buffer (100 mM TrisHC1 (pH 7.5), 0.9% NaCl, 0.1% [v/v] Tween 20, 1% bovine
serum albumin, 0.5% gelatin) strips were cut and used to test
antibodies diluted in TTBS buffer. Detection was using I25 Ilabeled protein A and autoradiography. For immunoprecipitations
assays, antibodies were pre-bound to 25 /tl protein A Sepharose
and washed four times in NET 2 buffer (50 mM Tris-HCl (pH
7.5), 150 mM NaCl, 0.05% Nonidet P^0, 0.5 mM
dithiothreitol). 20 /tl reactions with or without 1 /tg of cold
ABPA3' RNA contained 15% in vivo 32P-labeled nuclear
extract, 30% TEA buffer, 3.2 mM MgCl2, lmM Dithiothreitol
and 2u//tl RNasin. They were incubated for 10 min at 0°C, added
to antibodies bound protein A Sepharose beads and then again
incubated for two hours at 4°C. After six washes in NET 2
buffer, bound material was submitted to proteinase K digestion.
Released RNAs were extracted, separated by electrophoresis in
10% polyacrylamide-urea gels in TBE buffer and finally detected
by autoradiography. The so-referred 'purified' Ul and U2
snRNPs were similarly immunoprecipitated. Immunoselection of
RNase T1 RNA fragments were in 20 /tl reactions as described
(28) starting from 1 X 106 cpm (cerenkov counting) ABPA3' T7
RNA. Electrophoresis of protected fragments was on prerun 20%
polyacrylamide-8 M urea gels in TBE buffer.
RESULTS
The RNA complementary to the 5' end of Ul snRNA
Previous work in our laboratory led to the conclusion that RNAs
containing a sequence complementary to the 5' terminus of
mammalian Ul snRNA (nt 1 — 11) are competent to
instantaneously form, at 0°C and in the absence of ATP, an
abundant and stable complex depending on the integrity of the
5' terminus of Ul snRNA sequence (28). This complex is
perfectly visible in non-denaturing gels under technical conditions
where Ul snRNP is neither detected in pre-splicing nor in splicing
complexes (31). Since the ABPA3' RNA only contains the
consensus 5' splice site among all sequences characteristic of premRNAs, we surmised that it could be a suitable substrate to now
determine whether U2 snRNP can contact already bound Ul
snRNP. The ABPA3' RNA has been described in a previous work
(28). Here the ABPA3' sequence was inserted into the HindlHEcoRl sites of the pSP 73 plasmid and RNA was transcribed
using T7 RNA polymerase (Figure 1).
An U1-U2 interaction revealed by monospecific antibodies
against U2 and Ul snRNPs
Northern blot analyses from retarding gels having revealed that
the Ul snRNP-depending complex formed is poorly separated
from endogenous snRNPs, we have based our investigation on
the use of antibodies. Should Ul - U 2 snRNP contacts mediated
by an RNA containing a 5' splice site exist, then an anti-(U2)
RNP antibody should precipitate Ul snRNP as well as, after
RNase Tl digestion, an RNA fragment corresponding to the
portion of sequence where Ul snRNP interacts. Reciprocally,
an anti-(Ul) RNP should be able to precipitate some U2 snRNP
in the presence of an RNA complementary to the 5' end of U1
snRNA. We have used three antibodies, one from a patient serum
(Go) we first listed as solely being of anti-(Ul) RNP specificity
(32), a second one called 'pick Ul - U 2 ' (33) and the anti-(U2)
RNP, from patient Ya serum, known to precipitate U2 snRNP
predominantly, a small amount of Ul snRNP except when
diluted, but neither U 4 - U 6 nor U5 snRNPs (34). A detailed
analysis including immunoprecipitations and immunoblots was
carried out to determine whether the precipitation of some U1
snRNP by the patient Ya serum could be or not an obstacle in
experiments aimed at deciphering Ul - U 2 interactions in crude
nuclear extracts. Figure 2A shows the RNAs that are precipitated
Nucleic Acids Research, Vol. 20, No. 14 3627
11
t
10
1
20
1
30
1
40
I
SO
i—
1
<
60
I
70
1
80
1
90
t
100
1
5'SB
Figure 1. Sequence of the ABPA3'RNA. The sequence has been numbered counting the cap as nucleotide 0. A segment refers to the RNAse Tl fragment protected
already identified (ref.28 and Figure 5). Box refers to the sequence complementary to the 5' end of Ul snRNA.
from 32P-labeled HeLa cells nuclear extracts by the patient Go
serum (lane 1), the patient Ya serum (lane 2) and pick Ul — U2
(lane 3). As expected, the patient Ya serum predominantly
precipitates U2 snRNA and some Ul, while 'pick U 1 - U 2 '
precipitates considerable amounts of both. Surprisingly, the first
listed anti-(Ul) RNP serum Go also precipitates some U2 snRNA
suggesting that nuclear extracts competent for splicing contain
some complexes where Ul and U2 snRNPs are already
interacting. Other immunoprecipitations were carried out using
a fraction from glycerol gradients depleted of all but U1 snRNP
(Materials and Methods) as source of antigen. This isolated Ul
snRNP was precipitated by the Go (lane 4) but not at all by the
Ya serum (lane 5) although being native since containing all of
the antigenic 70 Kd, A and C proteins (not shown).
It has been demonstrated previously that the U1 RNP-specific
A and the U2 RNP-specific B" proteins share at least one
common epitope (35) therefore possibly explaining why the serum
Ya precipitates a small amount of Ul snRNA in addition to U2
snRNA. However, the Ya serum identifies the U2 RNP-specific
A' and much more weakly the U2 RNP-specific B" proteins (ref.
34 and Figure 2B, lane 3). It does not recognizes the Ul snRNPspecific A protein from crude nuclear extracts (Figure 2B, lane
3) in contrast to what was observed by Mimori et al. (34) from
a Ul snRNP enriched sample. However, our finding that
'purified' Ul snRNP is not at all precipitated by serum Ya rather
agrees with our observation and provides evidence that the
presence of the Ul snRNP-specific A protein cannot explain why
serum Ya precipitates Ul snRNP from crude extracts.
Our immunoblots could lead to the conclusion that the sera
'pick Ul —U2' and Go share common specificities since both
recognize the Ul snRNP-specific 70 Kd and A proteins and what
is most likely the U2 snRNP-specific B" at least in the case of
pick 'Ul — U2' (Figure 2B, lanes 4, 5 respectively). However,
serum Go is unable to precipitate U2 snRNP either from a
glycerol gradient fraction mainly containing U2 snRNP or from
a mixture made of 'purified' Ul and U2 snRNPs (see below in
Figure 5) in contrast to pick Ul - U 2 (not shown). One can note
also that pick U1-U2 decorates another band below the Ul
snRNP-spcific 70 Kd protein (lane 5).
In brief, our results argue that the already described
coprecipitation of Ul and U2 snRNAs by serum Ya (34) cannot
be solely explained by the presence of a low level of anti-(Ul)
RNP antibodies or by common epitope sharing of A and B"
proteins (35). It seems also likely that some snRNP complexes
containing both Ul and U2 snRNAs could exist in nuclear extracts
or be created upon incubation. This is supported by two
independent results. The first one is that serum Go, described
as being of Ul snRNP specificity, also precipitates a small amount
of U2 from these nuclear extracts (fig. 2A, lane 1). The second
one is that the U2-specific monoclonal antibody 4G3 was, in our
/
v
Go
Ya
Pick U1-U2
SPLICING ECTEACT
PUROTn) Ul n S N P
+
+
+
+
+
+ + +
+ +
B
M 1 2 3 4 5
12 3 45
Figure 2. (A) Immunoprecipitation of in \ivo 32 P labeled snRNAs. Precipitates
analyzed for their snRNA content were either from a splicing nuclear extract
(lane 1 - 3 ) using the anti-(Ul) RNP serum (Go) (lane 1). the anti-(U2) RNP
serum (Ya) (lane 2), the anti-CUl,U2) RNP serum (pick U1-U2) (lane 3) or
from 'purified' Ul snRNP (Materials and Methods) using the anti-{Ul) RNP
serum (Go) (lane 4) and the anti-(U2) RNP serum (Ya) (lane 5). Lane M shows
snRNA markers (B) Immunoblots analyses. All nitrocellulose strips originate from
a single blot containing SDS-gel fractionated proteins from an Hela cells splicing
nuclear extract. They were probed with either one of the following antibodies:
The monoclonal anti-(Ul) RNP (2.73) (lane 1), the monoclonal anti-Sm (Y12)
(lane 2), the anti-(U2) RNP serum (Ya) (lane 3), the anti-{U 1) RNP serum (Go)
(lane 4) and the anti-(U 1 ,U2) RNP serum (pick U1 - U2) (lane 5) and then revealed
by II5I-labeled protein A and autoradiography.
hands, able to immunoprecipitate a significant amount of Ul
snRNP from splicing extracts (not shown). Both these findings
agree with previous results demonstrating that a fraction of U1
and U2 snRNPs are interacting in Xenopus laevis oocytes (36).
ABPA3' RNA induces coprecipitation of U2 and Ul snRNAs
Two aliquots of in vivo 32P-labeled nuclear extract were
incubated at 0°C for 15 min in the presence or the absence of
1 /tg of unlabeled ABPA3' RNA. Immunoprecipitations were then
performed using either non-diluted or diluted serum Ya under
the same conditions as in Figure 2A. The results are shown in
Figure 3. In the absence of ABPA3' RNA, serum Ya precipitates
U2 and trace amounts of Ul snRNAs (lane 2) as expected from
the above results. In the presence of ABPA3' RNA (lane 1), the
amount of Ul snRNA precipitated is clearly above the control
3628 Nucleic Acids Research, Vol. 20, No. 14
value (compare lane 1 with lane 2 in Figure 3A). Quantitative
estimations from several experiments using three dilutions of
serum Ya have revealed that the amount of precipitated Ul
snRNP is about increased three fold in the presence of ABPA3'
RNA, while that of precipitated U2 snRNP remains virtually
unchanged (see densitograms in Figure 3B). Such a result argues
that the coprecipitation of the two snRNPs is not due to the
B
Ya serum
dilution
A
1 1
0
Ya serum
25 25
ABPA3' 1 0 1
0
1
100
1/12
1/25
i
In
0
0«g)
presence of contaminating anti-(Ul) RNP in serum Ya, but rather
that Ul snRNP is precipitated through interaction with U2
snRNP.
As a further check on this U1/U2 snRNPs coprecipitation
induced by an RNA complementary to the 5' end of Ul snRNA,
we carried out the reciprocal experiment consisting in
immunoprecipitating snRNPs with anti-(Ul) RNP instead of anti(U2) antibodies. In this case, we have used Ul and U2 snRNPs
partially purified by glycerol gradient centrifugation as source
of antigen (Figure 4A), therefore avoiding the spontaneous
U1-U2 interaction seen above in Figure 2A. The results are
shown in Figure 4B. The monoclonal antibody 2.73 (lane l) and
serum Go (lane 2) precipitate purified Ul snRNP (the band below
Ul is Ul* (37) generated during immunoprecipitation). These
same antibodies also precipitate contaminating U1 from the U2
snRNP rich fraction but neither one precipitates U2 snRNA Qanes
3, 4). Similarly, the two antibodies only precipitate Ul snRNP
when purified Ul and U2 snRNPs are put together (lanes 5,6).
In contrast, they now precipitates Ul and a significant amount
of U2 snRNP upon addition of ABPA3' RNA Ganes 8, 9 and
quantitative estimations in Figure 4 Q . Therefore, this experiment
confirms what we have seen above using serum Ya. In contrast
to what was observed in lanes 1—6 (Figure 4B), Ul* does not
appear in the presence of ABPA3' RNA, signifying that most
if not all of the Ul snRNPs become protected from nuclease
degradation. Identical results were obtained using serum Ya
instead of anti-(Ul) RNP and 'purified Ul and U2 snRNPs as
source of antigen (not shown).
U2
n
Ul
12
ABPA3'
RNA
3 4
Figure 3. Coprecipitation of U2 and Ul snRNPs by serum Ya in the presence
of the ABPA3' RNA. (A) Immunoprecipitations were from the same splicing
nuclear extract as in Figure 2 (lane M) using 1 /il of 25-fold diluted anti-(U2)
RNP serum Ya (lanes 1, 2). Lanes 3, 4 show controls without antibody. 1 /tg
of unlabeled ABPA3' RNA was added to each of the assays corresponding to
lanes 1 and 3. (B) Densitometric analysis of the above and other assays using
different dillutions of serum Ya. Value 100 was arbitrarily given to the greatest
amount of immunoprecipitated U2 snRNA using non-diluted serum Ya. +/ —
refer to the presence or the absence of ABPA3' RNA in the immunoprecipitation
reaction.
U2 snRNP contacts Ul snRNP already bound to ABPA3'
RNA
The above assays clearly show that an RNA complementary to
the 5' end sequence of Ul snRNA induces coprecipitation of Ul
and U2 snRNPs by either one of the corresponding monospecific
2.73
B
ANTIBODY
I
Purified snRNPs
5
10
15
20
24
5'SS ABPA.V
U2
111
In
>
U2
U4
U5
116
Ul
i
ir
f + +
m*L1
U4 —
5SU5_
Figure
4-
Go
U2
>
>
M
I 1'2 snRNPs h\ anti-fl'l) RNP antihod:-
1 2 3 4 5 6 7 8 9
ABPA.VR^
AIIPA3'
RNA
Go
1
II
.
ait was
carried out using fractions enriched in either Ul or U2 snRNPs which were obtained by fractionation of a 32P-labeled nuclear extract through a 10% glycerol gradient
(Materials and Methods). (A) shows the snRNA distribution throughout the gradient. Note that the gel was weakly exposed explaining why U4, U5 and U6 snRNAs
are poorly visible, lane M shows total RNAs from the extract). The fractions corresponding to lanes 6 - 9 and 11-14 were pooled, concentrated (Materials and
Methods) and used as source of 'purified' Ul and 'purified' U2 snRNPs, respectively. Note that the 'purified' Ul pool contains trace amounts of U2 snRNP and
vice versa. (B) shows immunoprecipitated snRNAs by the monoclonal anti-{Ul) RNP (2.73) antibody from 'purified' Ul snRNP (lane 1), 'purified' U2 snRNP
(lane 3), and a mixture of both reflecting the U1/U2 ratio existing in nuclear extracts in the presence (lane 8) or the absence (lane 5) of 1 ng of unlabeled ABPA3'
RNA). Lanes 2, 4, 6, 9 correspond to the same sources of antigen as lanes 1, 3, 5, 8 but theantHUl) RNP serum (Go) was used instead of the monoclonal anti-{Ul)
RNP (2.73) antibody. Lane M and lane 7 respectively refer to markers (total RNAs from the nuclear extract) and to an assay without antibody from a mixture
of the two particles also containing 1/ig of cold ABPA3' RNA. (C) shows a similar densitometric analysis as in Figure 3B of immunoprecipitated RNAs seen in
B lanes 5, 6 and 8, 9. Here, value 100 was given to the greatest amount of immunoprecipitated Ul snRNA for each antibody. For calculation, densitometric values
corresponding to Ul* were added to those corresponding to Ul.
Nucleic Acids Research, Vol. 20, No. 14 3629
antibodies. However, it remains to be determined whether U2
snRNP contacts Ul bound to the ABPA3' RNA through the 5'
end of its snRNA, or independently binds to the RNA. To
distinguish between these two possibilities, we have carried out
RNase Tl irnrnunoprecipitation assays in order to map the snRNP
binding sites (38). In addition to serum Go and Ya (Figure 5,
lanes 1—3, 13 and 5 - 7 , respectively), we have used the 2.73
monoclonal anti-RNP (lanes 9-11) and anti-cap antibodies (lanes
14-16) able to precipitate all snRNPs. ^P-labeled ABPA3'
RNA was incubated during 30 min at 0°C without ATP in either
standard (lanes 1, 5, 9, 13, 14), Ul snRNA-cleaved (lanes 2,
6, 10-15) or U2 snRNA-cleaved (lanes 3, 7, 11, 16) nuclear
extracts (see Materials and Methods for site-directed cleavage
of Ul and U2 snRNAs) in the presence of one of the above
antibodies and RNase Tl. In all cases, electrophoresis of
immunoprecipitated material through a 20% sequencing gel
essentially revealed the presence of one 17 nt. fragment
(designated A in Figure 1) that subsequent secondary RNase Tl
analysis (not shown, see in ref. 28) identified as being derived
from the consensus 5' splice site contained in ABPA3' RNA.
In the absence of additional protected fragment using serum Ya
and anticap antibodies and since the presence of the 17 nt.
fragment depends on the Ul snRNA 5' terminus integrity
whatever the antibody used, we conclude that U2 snRNP does
not interact directly with the ABPA3' RNA but rather with the
already bound Ul snRNP. This was confirmed by the absence
of any precipitated fragment when the ABPA3' RNA was
replaced by an RNA lacking the Ul snRNA complementary
sequence, for example an RNA only containing the human /3globin polypyrimidin tract 3' splice site sequence (not shown).
One can note in addition that the 5' terminus of U2 snRNA is
Go
Ya
2.73
Go anti Cap
d#S#bWl^o# V
19
>j
13
11
1 2 3 4 5 6 7 8 9101112M
10
9
8
1314 1516 17 M
Figure 5. Analysis of RNase Tl protected fragments from ABPA3' RNA
immunoprecipitated by diverse antibodies. 32 P labeled ABPA3' RNA was
incubated at 0°C without ATP in standard (lanes 1, 5, 9, 13-14), Ul snRNA
cleaved (lanes 2, 6, 10, 15) or U2 snRNA cleaved (lanes 3, 7, 11, 16) nuclear
extracts in the presence of one of the following antibodies: monoclonal antHUl)
RNP (Go) (lanes 1 - 3 and 13), anti-{U2) RNP (Ya) (lanes 5 - 7 ) , antHUl) RNP
(2,73) (lane 9-11) and anti-2,2,7 trimethyl guanosine (lanes 14-16) antibodies.
In all cases the amount of antibody was sufficient to quantitatively precipitate
the snRNPs in the reaction. In all assays where the anti-(U2) RNP antibody was
used, the totality of the precipitated 32P-labeled material was layered on the gel.
In all other cases, only 1/6 of precipitated material was layered in order to obtain
the same amount of counts (Cerenkov counting) in all lanes. Products from RNAse
Tl digestion of the ABPA3' RNA were used as markers (lane M) and assays
without antibody were carried out as a control (lanes 4, 8, 12, 17). Protected
fragments were processed and separated as described in Materials and Methods.
not involved in this Ul — U2 snRNPs interaction since cleavage
of this sequence has no effect on the precipitation whatever the
antibody used (Figure 5, lanes 3, 7, 11, 16).
DISCUSSION
We have exploited the stable binding of Ul snRNP to an RNA
containing the complementary sequence of the 5' terminus of Ul
snRNA to investigate the possibility that U2 snRNP may interact
with bound Ul snRNP independently of its own binding to the
branchpoint sequence of pre-mRNAs. With the use of
monospecific antibodies, we have obtained evidence that the
presence of this antisense RNA, either in a crude nuclear extract
or in an artificial mixture enriched in Ul and U2 snRNPs, leads
to the precipitation of a significant amount of interacting Ul and
U2 snRNPs. This is in addition to free and bound Ul snRNP,
in the case of anti-<Ul) RNP antibody, and free U2 snRNP in
the case of anti-(U2) RNP. As a matter of fact, our finding that
the precipitation of protected RNA by anti-(U2) RNP and anticap antibodies requires Ul snRNA integrity, as well as the
observation that U2 snRNP cannot contact an RNA lacking the
sequence complementary to the 5' terminus of Ul snRNA, argue
that U2 snRNP interacts with bound Ul rather than directly with
another region of the RNA.
How this snRNP-snRNP interaction arises? First, it was more
easy to detect it using such a synthetic RNA instead of a true
pre-mRNA. Indeed, the presence of a branchpoint-3' splice site
could lead to U2 snRNP binding in this region, even in the
absence of ATP, thus rendering difficult the results to interpretate.
Second, this interaction appears as being different from the
spontaneous Ul - U 2 contact reported in earlier studies (36, 39)
and also seen here in splicing extracts (Figure 2A). Indeed, it
results of the presence of a third partner. Assuming that Ul
snRNP binds to this RNA as part of a multi-snRNP particle
containing Ul and U2 snRNPs, it seems difficult to explain why
this complex would become more accessible to antibodies.
However, we cannot completely rule out the possibility that the
spontaneous U 1 - U 2 snRNPs contact might be due to
endogeneous RNAs containing 5' splice site sequences. The
simpler explanation most likely is that some conformational
changes have occurred in bound Ul snRNP, therefore allowing
U2 snRNP to enter the 5' splice site-Ul snRNP complex, for
example through protein-protein interaction.
Is this snRNP-snRNP interaction induced by such an artificial
RNA representative of an event arising at very early times in
the spliceosome assembly pathway? Recently, regarding RNase
Tl-immunoprecipitation experiments similar to that we have
performed, it has been reported that an anti-(U2) RNP antibody
precipitates a small amount of Tl fragment corresponding to the
Ul snRNP binding site (40). This was explained either by weak
interactions between U2 snRNP and a complex bound to the 5'
splice site or by a low-level contamination of anti-(Ul) RNP
antibody in the anti-(U2) RNP serum. The results described here
favour the former hypothesis. Another interesting finding is that
the so-designated E pre-spliceosome complex which forms in the
absence of ATP contains significant levels of U2 snRNP (41).
What we describe here perfectly agrees with this observation.
In other words, E complex could be formed with an RNA
containing a 5' splice site but lacking a branchpoint 3' splice site.
It has been also reported that Ul snRNP interacts with the premRNA branchsite region at an early stage of spliceosome
assembly (24—26) and, more recently, that there is a requirement
3630 Nucleic Acids Research, Vol. 20, No. 14
for Ul snRNP to allow stable binding of U2 snRNP at the
branchpoint sequence (27). As this function of Ul snRNP is
independent of either the 5' terminus of Ul snRNA or the
presence of a 5' splice site on the pre-mRNA, it has been
proposed that an Ul - U 2 snRNP interaction is possibly involved.
Finally, it appears that the results reported by Michaud and Reed
(40), those presented in this paper and those reported by Barabino
et al. (27) can be combined to add new details to the scheme
of the spliceosome assembly pathway. Indeed, it appears quite
plausible that the so-called pre-spliceosome complex, in which
the 5' and 3' splice sites most likely are juxtaposed, thus
prefiguring the formation of the lariat intermediate, might be
preceded by either one of two different E pre-spliceosome
complexes, one (El) with interacting U2 and Ul snRNPs at the
branchpoint sequence and a second one (E2) with interacting Ul
and U2 snRNPs at the 5' splice site.
ACKNOWLEDGEMENTS
Thanks are due to R. Dietz for excellent technical assistance.
This work was supported by grants from Centre National de la
Recherche Scientifique (UA 1191), Association pour la
Recherche contre le Cancer (contrat 6952), Ligue Nationale
contre le Cancer, and Fondation pour la Recherche M6dicale.
M.C. Daugeron is supported by a fellowship from the Ligue
Nationale contre le Cancer.
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