RPL30 regulation of splicing reveals distinct roles for Cbp80 in U1 and U2 snRNP cotranscriptional recruitment
Pre-mRNA splicing is catalyzed by the spliceosome, and its control is essential for correct gene expression. While splicing repressors typically interfere with transcript recognition by spliceosomal components, the yeast protein L30 blocks spliceosomal rearrangements required for the engagement of U2 snRNP (small ribonucleoprotein particle) to its own transcript RPL30. Using a mutation in the RPL30 binding site that disrupts this repression, we have taken a genetic approach to reveal that regulation of splicing is restored in this mutant by deletion of the cap-binding complex (CBC) component Cbp80. Indeed, our data indicate that Cbp80 plays distinct roles in the recognition of the intron by U1 and U2 snRNP. It promotes the initial 5' splice site recognition by U1 and, independently, facilitates U2 recruitment, depending on sequences located in the vicinity of the 5' splice site. These results reveal a novel function for CBC in splicing and imply that these molecular events can be the target of a splicing regulator.
RPL30 regulation of splicing reveals distinct
roles for Cbp80 in U1 and U2 snRNP
and JOSEP VILARDELL
Centre de Regulacio
mica, 08003 Barcelona, Spain
Molecular Biology Institute of Barcelona (IBMB/CSIC), 08028 Barcelona, Spain
Pre-mRNA splicing is catalyzed by the spliceosome, and its control is essential for correct gene expression. While splicing
repressors typically interfere with transcript recognition by spliceosomal components, the yeast protein L30 blocks spliceosomal
rearrangements required for the engagement of U2 snRNP (small ribonucleoprotein particle) to its own transcript RPL30.Using
amutationintheRPL30 binding site that disrupts this repression, we have taken a genetic approach to reveal that regulation of
splicing is restored in this mutant by deletion of the cap-binding complex (CBC) component Cbp80. Indeed, our data indicate that
Cbp80 plays distinct roles in the recognition of the intron by U1 and U2 snRNP. It promotes the initial 59 splice site recognition by
U1 and, independently, facilitates U2 recruitment, depending on sequences located in the vicinity of the 59 splice site. These
results reveal a novel function for CBC in splicing and imply that these molecular events can be the target of a splicing regulator.
Keywords: regulated splicing; Cbp80; 59SS; U1 snRNP/U2 snRNP
Most eukaryotic pre-mRNAs need to be spliced before
being translated. During pre-mRNA splicing, intervening
sequences (introns) are precisely removed and the adjacent
sequences (exons) are spliced together. This process takes
place in the spliceosome, a large complex ribonucleoprotein
particle (RNP) (for review, see Wahl et al. 2009). Assembly of
the spliceosome starts cotranscriptionally with the recogni-
tion of the 59 splice site (59SS) by the U1 small nuclear RNP
(snRNP), committing the transcript to splicing. This can be
assisted by the cap-binding complex (CBC), an heterodimer
of the factors Cbp20 and Cbp80 (Izaurralde et al. 1994; Fortes
et al. 1999b). Subsequently, the 39 end of the intron, in-
cluding the branch site (BS) and the 39 splice site (39SS), is
identified by the factors BBP and Mud2 in budding yeast, and
SF1 and U2AF in metazoans. An interaction between these
components and U1 snRNP has been shown in vitro in this
complex (Abovich and Rosbash 1997; Kent and MacMillan
2002), known as commitment complex or CC (complex E in
metazoans). In the next step, a poorly understood remodel-
ing of the CC takes place leading to the association of U2
snRNP with the BS, to form the pre-spliceosome or complex
A (Parker et al. 1987; Wu and Manley 1989; Zhuang and
Weiner 1989). Next, the pre-spliceosome is further remod-
eled and the tri-snRNP U4/U6.U5 engages in the nascent
particle (complex B). Subsequent rearrangements involving
the displacement of U1 and U4 snRNP lead to the formation
of a catalytically active spliceosome (complex C) (Wahl et al.
2009). In addition to its role in the formation of the CC, the
CBC has also been shown to promote association of U4/
U6.U5 to the nascent spliceosome (O’Mullane and Eperon
1998; Staley and Guthrie 1999; Gornemann et al. 2005).
Spliceosome assembly is tightly regulated at multiple levels
(Graveley 2000; Dreyfuss et al. 2002; Hertel and Graveley
2005; House and Lynch 2008), and work in Saccharomyces
cerevisiae has provided relevant data on splicing mechanisms
and regulation (Brow 2002; Meyer and Vilardell 2009). One
example is the essential RPL30 gene, encoding the ribosomal
Western General Hospital (MRC), Crewe Road,
EH4 2XU Edinburgh, Scotland, UK;
de Recerca i Estudis
Avancxats (ICREA) and IBMB, Baldiri Reixac 10-12, 08028 Barcelona,
Reprint requests to: Josep Vilardell, Institut Catala
de Recerca i Estudis
Avancxats (ICREA) and Molecular Biology Institute of Barcelona (IBMB),
Baldiri Reixac 10-12, 08028 Barcelona, Spain; e-mail: josep.vilardell@
ibmb.csic.es; fax: +34-93-4034979.
Article published online ahead of print. Article and publication date are
RNA (2010), 16:2033–2041. Published by Cold Spring Harbor Laboratory Press. 2033
protein L30 which, when in excess, binds
to itsowntranscriptand stalls spliceosome
assembly. L30 interacts with a kink-turn
structure (Klein et al. 2001) that mimics
the L30 rRNA binding site (Vilardell et al.
2000). This prevents association of U2
snRNP with the BS by a distinct mecha-
nism likely to involve an interference with
conformational changes that occur during
spliceosome assembly (Macı
as et al. 2008).
Here we follow a genetic approach to ad-
dress the molecular mechanisms involved
in this regulation. Taking advantage of
silent RPL30 mutants that affect splicing
regulation by L30, we have sought muta-
tions in trans that restore control of
splicing. We have identified multiple mu-
tations in the CBC component Cbp80,
indicating that this factor has a wider role
in spliceosome assembly than previously
assumed. This includes a function in U2
recruitment that is the target of a splicing
C9 in RPL30 is required by L30
to repress U2 snRNP recruitment
To dissect mechanisms involved in the
repression of U2 snRNP recruitment by
L30, we undertook a genetic approach
based on RPL30 mutant transcripts
known to abolish control of splicing
(Vilardell and Warner 1994). Mutations
in RPL30 used here, localized in the kink-
turn motif recognized by L30, are shown
in Figure 1A. C9U perturbs a helix involved in the kink-turn,
and it is predicted to weaken it. 5A reduces the large loop
(positions 17–50, Fig. 1A), not required for L30 binding
(Chao and Williamson 2004), to five adenines, thus likely
stabilizing the kink-turn. +12 combines 5A with moving the
59SS 12 nucleotides (nt) downstream. We have previously
shown that in this transcript L30 binding and splicing are
compatible, due to the added space between L30 and the
as et al. 2008), and we use this RNA as a control
for the experiments described below. As expected, C9U
disrupts L30 binding in vitro (Fig. 1B, lanes 7–12; Vilardell
et al. 2000), 5A shows more affinity to L30 (lanes 12–18), and,
importantly, the combination 5A C9U binds L30 as well
To determine the effect of 5A C9U on splicing regulation
in vivo we used the LCUP1 reporter system, based on fusing
RPL30 exon 1 and intron to the CUP1 ORF (Vilardell and
Warner 1997). Northern analyses (Fig. 1C) indicate that
splicing of wt LCUP1 transcripts is regulated by L30 (lane 5),
while C9U mutants fail to be regulated (lane 6). LCUP1 5A
transcripts are highly repressed under excess L30 (lane 3 vs.
1), consistent with increased affinity to L30. Interestingly,
regulation of splicing by L30 is not restored in LCUP1 5A
C9U transcripts (lane 4 vs. 2). We conclude that both the
stability and the sequence of the RPL30 kink-turn are im-
portant for regulation, and that the C9U mutation abolishes
regulation even of transcripts in which the kink-turn is stably
bound by L30.
There are two alternate possibilities to explain the C9U
phenotype of lack of repression. Either L30 is dislodged
during intron recognition or it remains associated with the
transcript but fails to repress U2 snRNP recruitment. To
distinguish between them, we determined the levels of co-
immunoprecipitation of L30 with U1 and U2 snRNAs in
presence of 5A C9U transcripts. We used the +12 RNA as
a positive control, where splicing and L30 binding are
FIGURE 1. L30 binds, but does not repress, the RPL30 5A C9U transcript. (A) Schematic
representation of the RPL30 kink-turn bound by L30, wt, and mutant versions, as indicated
(SS: splice site, the AUG is at the end of the first exon). Numbers refer to the start of
transcription. (B) Effect of mutations in RPL30 on L30 binding. RPL30 transcripts (0.5 pmol,
nt 1–123) were incubated with buffer (lanes 1,7,13,19) or with increasing amounts of MBP:L30
(50, 100, 200, 500, and 1000 ng; lanes 2–6, 8–12, 14–18, and 20–24, respectively). Transcripts
were wt (lanes 1–6), C9U (lanes 7–12), 5A (lanes 13–18), and 5A-C9U (lanes 19–24). Reactions
were analyzed in a 6% acrylamide gel. (C) Both C9U and 5A C9U transcripts fail to accumulate
pre-mRNA under conditions of L30 excess. W303 (lanes 1,2) or yJV25 (producing excess L30,
lanes 3–6) cells were transformed with pLCUP plasmids (schematized on the bottom) bearing
the indicated mutations (top). RNA was extracted, subjected to Northern analysis, and probed
with LCUP sequences (D) snRNP coimmunoprecipitation with L30. RPL30 transcripts (nt
1–347) were incubated under splicing conditions with MBP:L30. Reactions were immuno-
precipitated with anti-MBP, and pelleted RNA was subjected to Northern analysis to detect
RPL30, U1 and U2 snRNA, as indicated. Lane 1, RPL30 +12; lane 2, RPL30 5A C9U; lane 3,
RPL30; lane 4, no transcript added; lane 5, no antibody added; lane 6, 1% of the input.
Bragulat et al.
2034 RNA, Vol. 16, No. 10
as et al. 2008). Figure 1D shows similar
levels of U1 coimmunoprecipitation in wt (lane 4) and 5A
C9U transcripts (lane 5), consistent with the initial intron
recognition by U1 snRNP being compatible with L30
binding. There is lack of U2 coimmunoprecipitation in both
transcripts, but not +12 (lane 6). This indicates repression
of splicing in wt RNA (Macı
as et al. 2008); however, since
RPL30 5A C9U is spliced even in presence of L30; lack of U2
coimmunoprecipitation is consistent with L30 removal
during U2 association with RPL30. Accordingly, we detect
U1 cross-linking with the 5A C9U 59SS, as well as association
of this intron with BBP, under repressive conditions (Supple-
mental Fig. S1). We conclude that a C at position 9 is required
for repression of splicing by L30, and that C9U blocks re-
pression of splicing after intron recognition by U1. Thus, we
decided to search for mutations that restore regulation of
splicing by L30 to transcripts with this mutation.
Screen for regulation of splicing of C9U transcripts
Our genetic screen is based on the CUP1 -based reporter
LCUPIF, in which Cup1 protein can only be expressed from
unspliced RNA (Fig. 2A); thus, LCUPIF splicing repression
by L30 is expected to produce increased copper tolerance.
Accordingly, LCUPIF confers copper resistance (>0.2 mM)
to a strain engineered to produce excess L30 (yJV25), while
LCUPIF C9U does not (Fig. 2B). Therefore, we anticipated
that cells with the LCUPIF C9U reporter would grow in
copper (>0.2 mM) if they bore a mutation-enhancing L30
repression of splicing. To select such mutants, yJV25 cells
carrying LCUPIF C9U were UV-irradiated and selected on
0.3 mM Cu
(see the diagram in Fig. 2C and Materials and
Methods), and six mutants showed splicing repression of this
reporter (Fig. 2D, lanes 3–8). These mutants, called SLR
Suppressors of Lack of Repression by L30), all exhibit
tolerance with LCUPIF C9U (Supplemental
Fig. S2). To test more specifically the effects on splicing
repression of our mutants we used the LCUP 5A C9U
reporter (Fig. 2A), not repressed in wt cells, but expected to
be repressed in SLR mutants. Complementation of slr with the
wt allele should restore full splicing of the 5A C9U reporter,
allowing selection by increased copper tolerance (as schema-
tized in Fig. 2C). Using this approach, a YCp50 plasmid
containing the CBP80 gene was found to restore wt levels of
copper tolerance to SLR4, SLR5, and SLR7 (Supplemental Fig.
S3). Northern analyses further verified that in these cells the
repression of splicing by L30 is similar to that in wt (Fig. 2E,
even lanes). We determined that the genomic copy of CBP80
includes the mutation L82Stop in SLR4, L157P in SLR5, and
D291Stop in SLR7 (Supplemental Fig. S4).
Deletion of CBP80 suppresses the requ irement
for C9 by L30
The isolation of two cbp80 alleles with premature Stop
codons prompted us to verify the Cbp80 levels in the
L157P mutant (SLR5). Western blot analyses indicate that
they are drastically reduced by this mutation (Fig. 3A),
suggesting that deletion of CBP80 in yJV25 could produce
an SLR phenotype. To assess this possibility we determined
that the Cu
tolerance of yJV25 cbp80D cells, bearing a LCUP
5A C9U reporter, was indistinguishable from that of SLR5
(Fig. 3B), and we verified by Northern analysis that in cbp80D
cells splicing of this reporter was repressed by L30 (Fig. 3C).
To verify that this effect is not due to a general decrease in
splicing efficiency in this intron, we analyzed its splicing and
regulation by L30 in cbp80D cells, using the pLGFP reporter
(based on pLCUP, with the ORF of CUP1 replaced by GFP)
as et al. 2008). Our results show that splicing of the
C9U reporter (insensitive to L30) is not affected by deletion
of Cbp80 (Fig. 3D upper panel, lanes 3,4), indicating that the
RPL30 intron is efficiently spliced in absence of Cbp80. In
addition, splicing of the 5A C9U RNA, but not that of C9U,
becomes repressed by excess L30 in cbp80D cells (Fig. 3D,
lower panel, lane 6 vs. 4). Therefore we conclude that absence
FIGURE 2. (Legend on next page)
Role of Cpb80 in U2 snRNP recruitment
of functional Cbp80 is the cause of augmented splicing
regulation by L30 on transcripts bound by L30. This is
also consistent with the increased pre-mRNA levels in
cbp80D in absence of additional L30 (endogenous L30 lev-
els suffice to trigger some repression; Fig. 3D, upper panel,
lane 2). We also verified that both splicing of C9U and splic-
ing regulation of wt transcripts were effective in cbp80D,
cbp20D, and cbp80D cbp20D (cbcD) strains (Supplemental
To test whether our findings from the genetic screen could
be confirmed in vitro, RPL30 transcripts were incubated with
extracts from either wt or cbp80D cells, supplemented with
recombinant L30. Our results (Fig. 4) show that, while overall
splicing activity is reduced in cbp80D extracts (Fig. 4B), 5A
C9U transcripts are specifically repressed by L30 in these
extracts (cf. lanes 8,9 from panels A and B). Importantly,
addition of recombinant Cbp80 restores splicing of 5A C9U
transcripts repressed by L30 (Fig. 4B, lane 10). These data
provide further support to the interaction between Cbp80
and repression of splicing by L30 and prompted us to analyze
the molecular bases for this interaction.
Effects of Cbp80 deletion on cotranscriptional
spliceosome assembly and L30 regulation
During control of splicing by L30 U2 recruitment is abol-
ished and U1 association with the intron becomes stabilized
as et al. 2008). Our data, showing enhancement of L30
repression by deletion of Cbp80, raise the possibility of a
novel role for CBC on U2 recruitment. Thus, we decided to
monitor cotranscriptional splicing regulation by L30 in wt
and cbp80D cells. For this we used the RPL30-LacZ reporter,
based on RPL30 with the LacZ ORF as the second exon. This
provides a convenient tool to assess cotranscriptional spli-
ceosome assembly and its regulation by ChIP (Macı
as et al.
FIGURE 2. Screen for synthetic enhancers of L30 repression of
splicing. (A) Reporter plasmids based on the fusion between RPL30
exon 1 and intron with the CUP1 ORF. LCUPIF transcripts (left)
produce Cup1 protein only when unspliced, while LCUP RNAs (right)
need to be spliced to encode the protein. (B) Phenotype, identified as
growth in medium-containing copper, of cells with constitutive excess
of L30 (yJV25) and transformed with either pLCUPIF (upper panel) or
pLCUP 5A (bottom panel). Under repression conditions (‘‘+’’ rows),
pLCUPIF confers copper resistance while pLCUP-5A does not. When
L30 repression is abolished by the C9U mutation (‘‘’’ rows), pLCUP
confers tolerance while pLCUPIF does not. Serial one-fifth dilutions
were spotted in each case. (C) Screen strategy to select mutations that
restore inhibition of splicing by L30 on a C9U transcript. Correspond-
ing Northern analyses are shown in panels D and E.StrainyJV25with
the plasmid pLCUPIF-C9U (Cu-sensitive) was UV-irradiated and SLR
mutants were selected on plates containing 0.3 mM copper. Colonies
showing LCUPIF C9U pre-mRNA accumulation (panel D) were cured
of the plasmid and transformed with pLCUP 5A C9U, rendering them
Cu sensitive again because of increased repression, unless the slr
mutation is complemented or suppressed. Thus, cells were transformed
with a YCp50-based wt genomic library and the transformants selected
on 0.7 mM copper, and pCBP80 was identified (panel E). (D)Northern
analysis of RNA from SLR mutant cells transformed with pLCUPIF
C9U (lanes 3–8). As controls, RNA extracted from yJV25 cells
harboring pLCUPIF wt (lane 1) or C9U (lane 2)wereloadedinthe
same gel. Precursor (p) and mature (m) LCUPIF transcripts are
indicated. (E) Northern analysis of RNA extracted from SLR4, SLR5,
and SLR7 (lanes 3–8) and yJV25 cells (lanes 1,2), transformed with
pLCUP 5A C9U alone (odd lanes), or plus pCBP80 (even lanes).
Precursor (p) and mature (m) LCUP transcripts are indicated. In D and
E U3 was used as loading control.
FIGURE 3. Deletion of CBP80 is synthetic with L30 repression
of splicing. (A) Decreased levels of Cbp80-TAP in SLR5 mutants.
Cbp80 protein was TAP-tagged at the C terminus in yJV25 (wt, lane 1),
SLR5 (lane 2), and cbp20D (Y02074). Extracts were subjected to
Western analyses, as indicated. Tubulin was used as loading control.
(B)DeletionofCPB80 produces the same phenotype as that of SLR5.
One-fifth serial dilutions of wt (yJV25, top), cbp80D (yJV35, middle),
and SLR5 (bottom) cells transformed with pLCUP 5A C9T were spotted
on copper-containing media, as indicated. Copper sensitivity indicates
splicing repression of the LCUP 5A C9U transcript (C). Deletion of
CBP80 leads to repression of LCUP 5A C9U splicing by L30. Northern
analysis of RNA from wt (yJV25, lane 1), SLR5 (lane 2), and cbp80D
(lane 3) cells. Positions of precursor (p) and mature (m) LGFP 5A C9U
are indicated on the right.(D) Splicing efficiency and repression by L30
of the RPL30 intron in wt and cbp80D cells, with or without excess L30.
Northern analysis of RNA from either wt (even lanes) or cbp80D cells
(odd lanes) transformed with the pLGFP reporter plasmid, as indicated
at the top. Samples in the bottom panel are from cells under excess L30
(pMB73). Positions of precursor (p) and mature (m) LGFP are indi-
cated on the left. pLGFP contains the GFP ORF instead of Cup1 in
pLCUP (Vilardell and Warner 1997). pMB73 encodes L30 without the
autoregulatory loop (Macı
as et al. 2008).
Bragulat et al.
2036 RNA, Vol. 16, No. 10
2008). The reporter was introduced in strains either pro-
ducing or not producing excess L30 (from plasmid pMB73)
as et al. 2008). ChIP analyses from these cells are shown
in Figure 5. We first verified that deletion of Cbp80 does not
lead to an increased cotranscriptional recruitment of L30,
which could otherwise be consistent with enhanced regula-
tion in cbp80D cells (Fig. 5B). Next we followed cotranscrip-
tional engagement of U1 snRNP on RPL30-LacZ (ChIP of
the U1 component Snu71-HTB; Macı
as et al. 2008). In
the absence of excess L30, the maximum U1 ChIP signal is
reduced in cbp80D cells, but it lingers toward the 39 end of the
gene (Fig. 5C,D, black lines). Under excess L30, ChIP of U1
shows a slight reduction and a similar persistence (Fig. 5D,
gray line), which can also be observed in wt cells (Fig. 5C,
gray). Cotranscriptional recruitment of U2 (ChIP of the U2
component Lea1-HTB) (Macı
as et al. 2008) in wt cells is
strongly repressed by excess L30 (Fig. 5E). In cbp80D cells, U2
recruitment is diminished, with a further signal reduction
under excess L30 (Fig. 5F). Taking into account the persis-
tence of the U1 ChIP profile and the low U2 recruitment
observed in cbp80D cells, our data suggest that in the RPL30
intron cotranscriptional engagement of U2 is dependent
on Cbp80. A persistence of U1 ChIP on the target gene has
also been described in cbcD cells (Gornemann et al. 2005).
However, in the same cells U2 ChIP on ECM33 resembles
that of wt (Gornemann et al. 2005). We examined this ap-
parent discrepancy by analyzing the cotranscriptional re-
cruitment of U2 snRNP on the ACT1 gene, which is not
affected by L30 overexpression, in a cbp80 D strain. Neither
deletion of CBP80 nor excess L30 have a significant effect on
cotranscriptional recruitment of either U1 or U2 snRNP on
the ACT1 intron (Supplemental Fig. S6). This is consistent
with previously published results (Gornemann et al. 2005)
and indicates that the role of Cbp80 in cotranscriptional
spliceosome assembly is transcript specific.
Cbp80 and 59 splice site sequences
impact U1 and U2 snRNP
The persistence of U1 on the RPL30
transcript in cbp80D cells (Fig. 5D) can
reflect a limited cotranscriptional recruit-
ment of U2 (Macı
as et al. 2008), suggest-
ing a role for Cbp80 in this recruitment.
To further dissect the contribution of
Cbp80 to U1 and U2 recruitments we
investigated whether sequence features in
RPL30 could play a specific role. A par-
ticular attribute of the RPL30 intron is the
evolutionarily conserved 59SS sequence,
GUCAGUAU, unique in yeast introns.
This sequence can form 7 base pairs with
U1, while the ACT1 intron, with GUAUG
UUC at its 59 end, has the potential to
form 5 (Fig. 6A) and produces U1 and U2
ChIPs more resilient to Cbp80 deletion (Supplemental Fig.
S6). Thus, we investigated the role of these sequences in
combination with Cbp80 on spliceosome assembly in the
context of the RPL30-LacZ transcript. Mutants GUauGUuc,
containing the first 8 nt of ACT1 (lowercase indicates changes
in wt RPL30 to ACT1), and GUauGUAU, with positions 3 and
4asinACT1, were compared with the wt version GUCAG
UAU. These mutations have been shown not to affect RPL30
splicing in wt cells (Supplemental Fig. S7; Macı
as et al. 2008).
The results (Fig. 6) can be summarized as follows.
In wt cells, U1 cotranscriptional recruitment is influenced
not only by the potential base-pairing between the intron and
U1, but also by the particular 59SS sequence (Fig. 6B–D,
black). Thus, GUauGUAU (Fig. 6C) produces a ChIP signal
more than three times higher than that of wt RPL30 (Fig. 6B).
This difference is striking considering that the potential to
base-pair with U1 is similar in both constructs. Consistent
with this, disrupting the potential base-pairing of U1 to
positions 7 and 8 (GUauGUuc) produces intermediate levels
of U1 ChIP (Fig. 6D), despite having less potential for base-
pairing with U1 than RPL30 59SS (Fig. 6B). This indicates
that AU at positions 3 and 4 facilitates U1 recruitment. The
corresponding U1 ChIPs in cbp80 D cells (gray lines) indicate
that Cbp80 is required to take full advantage of the increased
recruitment afforded by the extra base-pairing of U1 to
intronic positions 7 and 8. Thus, deletion of Cbp80 has
a greater effect on the intron with GUauGUAU than on the
one with GUauGUuc at the 59SS, to the point that both
reporters recruit similar levels of U1 in absence of Cbp80
(Fig. 6C,D, respectively, gray lines).
U2 cotranscriptional recruitment in wt cells (Fig. 6E–G,
black) shows efficiencies that do not necessarily correlate
with those of U1. While U2 associates better with a GUauG
UAU intron than with GUCAGUAU (Fig. 6F,E, respectively),
the improvement does not match that of U1. Moreover,
FIGURE 4. In vitro regulation of splicing of RPL30 5A C9U by L30 in the absence of Cpb80.
Synthetic RPL30 transcripts, indicated at the top, were incubated under splicing conditions
with wt (A) and cbp80D (B) extracts, supplemented with MBP:L30 or MBP:Cbp80, as indi-
cated. Upon completion of the reaction, RNA was extracted and analyzed by semiquantitative
RT-PCR. Bands corresponding to substrate and spliced RNA are shown. Amounts of RNA,
extracts, and recombinant protein were equivalent in all reactions.
Role of Cpb80 in U2 snRNP recruitment
GUauGUAU and GUauGUuc introns produce similar U2
ChIPs (Fig. 6F,G), despite having distinct U1 ChIPs. This
absence of correlation between U1 and U2 recruitments
persists in cbp80D cells (Fig. 6E–G, gray lines). Interestingly,
this is more marked in the GUauGUAU and GUauGUuc
introns (Fig. 6F,G). While their U1 ChIP profiles are com-
parable (Fig. 6C,D, gray), U2 recruitment to GUauGUuc
introns remains closer to that of the wt. These data indicate
that the role of Cbp80 in U2 recruitment becomes more
apparent in introns with increased base-pairing to U1,
consistent with the low recruitment of U2 in GUCAGUAU
introns in cbp80D cells (Fig. 6E, gray). We conclude that
Cbp80 has roles in U1 as well as U2 cotranscriptional re-
cruitment, and these become more evident when the in-
teraction between U1 and the intron is hyperstabilized. We
verified that the introduced mutations do not induce
alterations in pol II recruitment (Supplemental Fig. S8),
and we ascertained that the ChIPs on ACT1 remained similar
across the samples (Supplemental Fig. S9).
Our model predicts that reducing the affinity for U1 of
RPL30 should improve its splicing in cbp80D extracts, and to
assess this possibility we synthesized two transcripts: RPL30
5A C9U, corresponding to the wt intron, and RPL30 5A C9U
UC, with UC at intron positions 7 and 8 abolishing the
potential extra base-pairing to U1 (Fig. 6A). Our results show
that in wt extracts both RNAs splice (Fig. 6H), with 5A C9U
UC yielding more product (Fig. 6H, lanes 2,5). As expected,
they are both only marginally sensitive to L30 (Fig. 6H, lanes
3,6). In contrast, in cbp80D extracts (Fig. 6I) splicing of 5A
C9U is reduced to low levels (Fig. 6I, lane 2), while that of the
5A C9U UC remains active (Fig. 6I, lane 6). Both are repressed
by L30 (Fig. 6I, lanes 3,7), and addition of recombinant Cbp80
restores splicing to some extent. This effect is specific to RPL30
5A C9U introns (Supplemental Fig. S10).
Data on the control of splicing by L30 support a novel
strategy for regulation, based on interference with spliceo-
somal transitions during U2 snRNP association with the in-
as et al. 2008). In a genetic screen for mutations
that alter this regulation we have identified Cbp80. This
factor forms, in conjunction with Cbp20, the CBC, and is
linked to the recognition of the 59SS during both early and
late steps in spliceosome assembly (Gornemann et al. 2005,
and references therein). Yet our genetic analysis suggests an
additional role for Cbp80 in U2 recruitment, and we have
tested this hypothesis. Here we show that U2 snRNP re-
cruitment is assisted by Cbp80, is influenced by the sequence
next to the 59SS, and can be targeted by splicing regulatory
factors like L30.
A mutation in the exon 1 of RPL30 that restores
U2 snRNP recruitment requires Cbp80
We have determined that the C9U RPL30 mutation abolishes
regulation of splicing even under conditions where L30
binding is restored (Fig. 1C). This suggests that destabilizing
the RPL30 kink-turn allows the spliceosome to compete with
L30 binding. Consistent with this, we show that L30 remains
associated with the 5A C9U transcript during initial intron
recognition by U1 but it fails to coimmunoprecipitate with
U2 (Fig. 1D; Supplemental Fig. S1). A spliceosomal confor-
mational change is required for U2 binding (Abovich et al.
1994), and it is plausible that L30 fails to prevent this because
it is dislodged from the C9U mutant RNAs at this step. In
a genetic screen based on this model, we have found that
Cbp80 is required for the spliceosome to successfully
compete with L30 (Fig. 2E), but not to splice RPL30 under
nonrepressive conditions (Fig. 3D). This can be reproduced
FIGURE 5. Cotranscriptional U1 and U2 recruitments on the RPL30-
LacZ transcript in wt and cbp80D cells. Horizontal axes show the
distance in nucleotides from the start codon. Vertical axes indicate
the signal relative to that of the promoter (first primer pair, or PP1).
The black bar indicates intron position. The ChIP profiles correspond
to wt (panels C and E)orcbp80D cells (panels D and F), under normal
conditions (black lines) or under L30 excess (gray lines). (A) Scheme
showing the positions of the PCR primers used for the ChIP analyses
of the RPL30-LacZ intron, relative to the translation start. (B) ChIP
against L30 (L30-TAP). In both cases there is L30 excess (pMB73, see
Materials and Methods). Black line, wt cells; gray, cbp80D cells. In the
following panels, ChIP profiles of the indicated proteins are shown,
performed on RPL30-LacZ.(C,D) ChIP against U1 snRNP (Snu71-
HTB). (E,F) ChIP against U2 snRNP (Lea1-HTB).
Bragulat et al.
2038 RNA, Vol. 16, No. 10
in vitro (Fig. 4). These data suggest an additional function
of Cbp80, beyond its known roles on recruitment of U1 and
U6 (Gornemann et al. 2005). Therefore, to determine the
molecular basis for this, we have analyzed in detail U1 and U2
cotranscriptional spliceosome assembly in cbp80D cells.
Role of Cbp80 on U1 and U2 snRNP recruitment
Our results are consistent with a role for Cbp80 in cotran-
scriptional U2 recruitment, which is more evident when the
potential base-pairing between the intron and U1 snRNP is
unusually strong, as in RPL30. This ex-
plains the genetic interaction of Cbp80
with L30 regulation, as RPL30 is an ef-
fective U1 snRNP recruiter (Fig. 5; Tardiff
et al. 2006). Regarding this U1 recruit-
ment, comparison of ChIP profiles from
AUGUAU and GUCAGUAU introns
reveals a critical role for intronic se-
quences in the vicinity of the 59 splice site,
even when the number of potential base
pairs to U1 snRNA does not change (Fig.
6B,C). This is in agreement with previous
reports indicating that initial recognition
of an intron by U1 is not limited by base-
pairing, and that GU
AUGU could be
favored over GUCAGU by the U1 snRNP
factor U1-C (Du and Rosbash 2002).
Interestingly, however, there is no appar-
ent change in the splicing efficiency of
both transcripts, or in their regulation
(Supplemental Fig. S7; Macı
as et al. 2008).
The effective recruitment of U1 to the
RPL30 intron depends on both its se-
quence and Cbp80, since in cbp80D cells
U1 ChIP on GUAUGU
AU and GUAUG
UC introns are equivalent (Fig. 6B–D).
Thus, our results indicate that positions
3 and 4, and 7 and 8, have different effects
on U1 recruitment. They also reveal that
mostly dependent on Cbp80, in contrast
to that of positions 3 and 4. Remarkably,
our ChIP data indicate that U2 recruit-
ment does not always correlate with that
of U1, arguing that cotranscriptional as-
sociation of U2 does not simply follow U1
recruitment. Thus, while mutation of
AU to GUAUGUUC in cbp80D
cells does not have a significant effect on
U1 ChIP, there is a clear reduction in U2
ChIP on a GUAUGU
AU intron (Fig. 5E).
This suggests that in cbp80D cells hyper-
stabilized U1 binding interferes with U2
recruitment, and we have verified this
possibility in vitro by showing that a reduced potential for
base-pairing to U1 improves splicing of RPL30 substrates in
cbp80D extracts (Fig. 6I). This was surprising, as Cbp80
promotes U1 recruitment (Colot et al. 1996; Fortes et al.
1999b), and it could be expected that a lower affinity for U1 is
detrimental for splicing in cbp80D extracts. Yet, it does not,
suggesting a different limitation, more consistent with a func-
tion for Cbp80 in U2 recruitment, antagonized by the binding
of U1 to the intron. Significantly as well, L30 repression is
active in all5A C9U substrates (Fig. 6I; Supplemental Fig.S10),
consistent with our finding that repression in 5A C9U RNA is
FIGURE 6. Effect of mutations in the RPL30 intron and Cbp80 on cotranscriptional
recruitment of U1 and U2 on the RPL30-LacZ gene. Horizontal axes show the distance in
nucleotides from the start codon. Vertical axes indicate the signal relative to that of the
promoter (first primer pair, or PP1). The black bar indicates intron position. The ChIP profiles
correspond to wt (black lines) or cbp80D cells (gray lines). (A) Panels indicate different
intronic 59 ends, with a schematic representation of the possible base-pairing with U1 snRNA.
GUCAGUAU panels are based on data from Figure 5. (B–D) ChIP profiles of U1 snRNP
(Snu71-HTB). (E–G) ChIP profiles of U2 snRNP (Lea1-HTB). (H,I) In vitro splicing of the
RPL30 intron, either wt or with reduced affinity for U1 snRNA, in wt or cbp80D extracts.
Splicing reactions were set up and analyzed as in Figure 4, using wt extracts (H) or extracts
from cbp80D cells (I), supplemented with MBP:L30 or MBP:Cbp80, as indicated. In the UC
transcript, intron positions 6 and 7 (AU) have been mutated to UC, which cannot base-pair to
U1. Bands corresponding to substrate and spliced RNA are shown. Amounts of RNA, extracts,
and recombinant protein were equivalent in all reactions (see Materials and Methods).
Role of Cpb80 in U2 snRNP recruitment
not due to a general reduction in splicing activity. The
interference with U2 snRNP recruitment by hyperstabilized
intron binding to U1 in cbp80D cells evokes the blocking of
U4/U6.U5 tri-snRNP recruitment in prp28 cells (Staley and
Guthrie 1999), suggesting a U1 snRNP remodeling prior to U2
recruitment. There are genetic and molecular data supporting
this U1 remodeling. Genetic interactions between U1 snRNP,
the CBC, and the BS have been observed (Fortes et al. 1999a).
In addition, mutations in U1 predicted to weaken the in-
teraction with the 59SS allow for some U2 recruitment in the
absence of ATP (Liao et al. 1992). Furthermore, the structure
of the human U1 snRNP bound to the 59SS argue in favor of
this possibility as well, with the factor U1-C stabilizing the
interaction U1-59SS while being subjected to long-range
protein connections within the particle (Pomeranz Krummel
et al. 2009). Perhaps remodeling of the bridging between U1
snRNP and the BS affects the binding of U1 to the 59SS, an
interaction clearly influenced by CBC (Gornemann et al.
2005). In this context, the genetic interaction between
Cbp80 and L30 could be explained by their antagonistic roles
on spliceosome assembly (Supplemental Fig. S11). L30 in-
hibits U2 recruitment by stabilizing a particular conformation
of U1 on the intron, while Cbp80 promotes U2 recruitment
and a likely U1 remodeling. In a C9U mutant L30 binding may
not be stable enough to repress unless Cbp80 is absent. Thus,
Cbp80 could facilitate the conformational change that com-
petes with L30 binding or that L30 inhibits.
Cbp80 and regulated splicing
Given its multiple roles in RNA processing (including an
indirect effect on histone ubiquitination) (Hossain et al.
2009), deletion of Cbp80 can have diverse effects on different
transcripts, depending on when its function is more critical.
Thus, while early spliceosome events on ACT1 are not se-
verely affected in cbp80D cells (Supplemental Fig. S6),
microarray analyses (Clark et al. 2002) indicate strong
changes in ACT1 splicing in these cells, suggesting an effect
on later stages of splicing. Consistent with this, microarray
data from cbp80D cells correlate well with those from cells
with deletions of factors involved either in U2 recruitment
(Mud2/U2AF, Msl1/U2B) or U4/U6.U5 tri-snRNP function
(Snu66) (Fig. 2 in Clark et al. 2002). This is also in agreement
with the ChIP data showing a role for Cbp80 in early and late
stages of spliceosome assembly (Gornemann et al. 2005).
Furthermore, the same analyses indicate that some of the
most affected transcripts in cbp80D cells are subjected to
regulated splicing and do not have consensus branch site
sequences. One example is theYRA1 transcript, with highly
regulated splicing in a scheme that includes the link between
splicing and RNA export (Preker and Guthrie 2006; Dong
et al. 2007), processes both affected by CBC. Therefore, the
involvement of Cbp80 in steps involving the recognition of
the 39 end of the intron by U2 snRNP could be targeted as well
by several other strategies of regulation.
Our findings may have implications for mammalian
systems, with higher variability in splicing signals and more
regulated splicing than yeast. CBC is part of the first exon
definition complex (Berget 1995), and as such it may play
a relevant role in splicing decisions involving alternative
59 and 39 splicing sites in these introns, consistent with a pos-
sible role in alternative splicing (Raczynska et al. 2010). More-
over, as CBC has been shown to affect cotranscriptional events
downstream from the first exon (Wong et al. 2007; Fong et al.
2009), it is not inconceivable that the described modulation
of U1 and U2 cotranscriptional recruitment affects a variety
of splice site choices along mammalian transcripts.
MATERIALS AND METHODS
Yeast strains and plasmids
Yeast strains and plasmids used in this study are described in the
yJV25 cells containing pLCUPIF C9U were UV-irradiated and
selected on 0.3 mM copper. Splicing of the (refreshed) reporter
construct was verified in 46 colonies by Northern analyses, and
six SLR mutants were isolated. To identify the mutation, the
pLCUP5A-C9U reporter was used, which confers copper resis-
tance to wt cells but not to SLR mutants. Details can be found
in the Supplemental material.
Copper assays, Northern analyses, and in vitro assays
Northern and in vitro assays were performed as in Macı
as et al.
(2008). Gel-shifts were done as in Vilardell and Warner (1994).
Copper assays were done as in Konarska et al. (2006). RT-PCRs
were done as in Hossain et al. (2009), except that our primers
contained an M13 sequence tag to ensure specific amplification,
and products were analyzed on a 10% nondenaturing, 13 TBE
polyacrylamide gel. One nanogram of RNA and 0.5 mg of recom-
binant protein were used in the reactions. Probes are detailed
in the Supplemental material.
Chromatin immunoprecipitation and quantitative PCR assays
were performed as described in Macı
as et al. (2008), using the
same primer pairs. Error bars are based on three independent
biological replicas. RPL30-LacZ mutants described in Figure 6
were generated by in vivo gap-repair cloning, via cotransforma-
tion of pJV44 digested with Bam/Spe and a PCR fragment con-
taining the corresponding mutations in the RPL30 intron. Positive
colonies were sequenced for verification. (See the Supplemental
material for a list of used primers.)
Supplemental material can be found at http://www.rnajournal.org.
Bragulat et al.
2040 RNA, Vol. 16, No. 10
We are grateful to C. Query and J. Valca
rcel for discussions and
critiques to the manuscript, and to F. Azorı
n, F. Gebauer, R.
ndez, and J. Warner for comments on the manuscript. This
research has been supported by the MEC (BFU grants), an EU-
MC Contract (#510183), and Agaur. S.M. and M.M. are sup-
ported in part by an Agaur fellowship and J.V. has been supported
by a Ramo
n y Cajal/IP3 contract (MEC).
Received July 14, 2010; accepted July 19, 2010.
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Role of Cpb80 in U2 snRNP recruitment