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www.landesbioscience.com RNA Biology 563
RNA Biology 6:5, 563-574; November/December 2009; © 2009 Landes Bioscience
RESEARCH PAPER
*Correspondence to: Panagiota Kafasla; Email: pk303 @cam.ac.uk
Submitted: 06/01/09; Revised: 08/10/09; Accepted: 0 8/18/09
Previously published online: ww w.landesbioscience.com/journals/rnabiology/article/9861
Introduction
The cap (m7GpppN) structure added co-transcriptionally to
RNA polymerase II transcripts has been shown to influence many
aspects of RNA metabolism, including pre-mRNA splicing,1,2
3' end formation,3 expor t f ro m t he nu cl eu s,4-6 st ab il it y7 a nd tr an s-
lation.8 In the nucleus, the cap structure interacts with the pre-
dominantly nuclear cap-binding complex (CBC), a heterodimer
consisting of cap-binding proteins CBP20 (Mud13p in S. cerevi-
siae) and CBP80 (Sto1p or Gcr3p in S. cerevisiae).9,10 CBP20 is
highly conserved from yeast to human, whereas CBP80 is far less
c on s er v ed . CB P2 0 re c og n iz e s an d bi nd s ca p pe d R N A in c on ju n c-
tion with CBP80.11 CBC plays a direct role in precursor messen-
ger RNA (pre-mRNA) splicing, promoting the association of U1
small nuclear ribonucleoprotein particle (snRNP) with the cap-
proximal 5' splice site.2,12 In Saccharomyces cerevisiae, CBC inter-
acts with Snu56p, a yeast-specific component of the U1 snRNP
and a cbp20-∆ cbp80-∆ double mutant strain shows synthetic
lethality with SNU71, a gene encoding for another component
of the yeast U1 snRNP, Snu71p.13 Furthermore, CBC exits from
the nucleus to the cytoplasm together with the mRNA,4-6 where
it is thought to be replaced by the eIF4F complex.
Interaction of yeast eIF4G with spliceosome
components
Implications in pre-mRNA processing events
Panagiota Kafasla,1,3,* J. David Barrass,1 Elizabeth Thompson,1 Micheline Fromont-Racine,2 Alain Jacquier2 Jean D. Beggs1 and
Joe Lewis1,†
1Wellcome Trust Centre for Cell B iology; University of Edinburgh; Edinbur gh, UK; 2Genetique des Interacti ons Macromoleculaires; Institut Pasteur
(CNRS-URA2171); Pari s, France; 3Department of Biochemistr y; University of Camb ridge; Camb ridge, UK
†Current ad dress: Chemical Bio logy Core Facility; EMBL; Heide lberg, Germany
Key words: pre-mRNA processing, spliceosome, eIF4G, Prp11p, Snu71p
RESEARCH PAPER
In the cytoplasm, the effect of the cap structure on mRNA
translation is mediated by a trimeric complex termed eukaryotic
translation initiation factor 4F, eIF4F. eIF4F consists of the cap-
binding subunit eIF4E, eIF4G and the RNA-helicase eIF4A.14
eIF4G acts as a bridge between the cap structure and components
of the ribosomal initiation complex.14,15 In addition to eIF4E and
eIF4A, eIF4G interacts with the poly(A)-binding protein PABP
(Pab1p in yeast) facilitating the functional association of the
3' end of an mRNA with its 5' end to promote translation,16 while
the association between eIF4G and eIF4E markedly enhances
the binding of the latter to the cap structure.14 eIF4G and CBP80
are both characterized by the presence of the MIF4G domain, a
structural motif known to be present in many proteins involved
in RNA metabolism.17,18 In S. cerevisiae there are two function-
ally redundant in translation isoforms of eIF4G, encoded by the
genes TIF46 31 and TIF4632.19
A stable association of eIF4G with CBC detected in the
nucleus of human cells plays possibly a role in coupling R NA-
processing events in the nucleus with mRNA translation in the
cytoplasm.20 In mammalian cells, the interaction between eIF4G
and CBC is required for the “pioneering” round of translation
that leads to nonsense mediated decay (NMD). NMD is a
As evidenced from mammalian cells the eukar yotic translation initiation factor eIF4G has a putative role in nuclear RNA
metabolism. Here we investigate whether this role is conserved in the yeast Saccharomyces cerevisiae. Using a combina-
tion of in vitro and in vivo methods, we show that, similar to mammalian eIF4G, yeast eIF4G homologues, Tif4631p and
Tif4632p, are present both in the nucleus and the cytoplasm. We show that both eIF4G proteins interact efciently in
vitro with UsnRNP components of the splicing machinery. More specically, Tif4631p and Tif4632p interact efciently
with U1 snRNA in vitro. In addition, Tif4631p and Tif4632p associate with protein components of the splicing machinery,
namely Snu71p and Prp11p. To further delineate these interactions, we map the regions of Tif4631p and Tif4632p that are
important for the interaction with Prp11p and Snu71p and we show that addition of these regions to splicing reactions in
vitro has a dominant inhibitory effect. The observed interactions implicate eIF4G in aspects of pre-mRNA processing. In
support of this hypothesis, deletion of one of the eIF4G isoforms results in accumulation of un-spliced precursors for a
number of endogenous genes, in vivo. In conclusion these observations are suggestive of the involvement of yeast eIF4G
in pre-mRNA metabolism.
564 RNA Biology Volume 6 Issue 5
with spliceosomal snRNPs are suggestive of a role for eIF4G in
nuclear RNA-processing, perhaps in coupling splicing to other
nuclear events and to translation.20
In the present work we assess the role of yeast eIF4G proteins
in processing of RNA in the nucleus. We show the presence of
eIF4G in the yeast nucleus and identify nuclear components that
interact with eIF4G. We characterize in detail the interaction
of eIF4G with protein and RNA components of the yeast spli-
ceosome. We further investigate the possible role of yeast eIF4G
in pre-mRNA splicing in vitro, and we show that depletion of
one of the eIF4G homologues in vivo results in accumulation
of intron containing pre-mRNAs for a number of endogenous
genes. Our results suggest that yeast eIF4G has a role in pre-
mRNA processing in the nucleus.
Results
Subcellular localization of Tif4631p and Tif4632p. To determine
whether the yeast homologues of eIF4G, Tif4631p and Tif4632p,
are present in both the cytoplasm and the nucleus, similarly to
their human homologues, the proteins were expressed from their
native promoter as C-terminally TAP-tagged fusion proteins
and their localization was determined by indirect fluorescence
and confocal microscopy. Both Tif4631p-TAP and Tif4632p-
TAP were, as expected, found to be abundant in the cytoplasm
(Fig. 1A and B). In addition, TAP-tagged Tif4631p and to a
lesser extent Tif4632p could be both detected also in the nucleus,
as shown in Figure 1B, in comparison to C where the location of
the nuclei is indicated, and in the merged image A. This finding
prompted us to determine whether the yeast eIF4G homologues
can interact with components of the splicing machinery, similar
to the situation observed in human extracts.20
Interaction of Tif4631p and Tif4632p with spliceosomal
snR NPs. Whole cell extracts were made from strains expressing
either Tif4631p-TAP or Tif4632p-TAP and the tagged proteins
were precipitated using protein A Sepharose. An isogenic wild
type strain was used as a negative control. Co-precipitated RNAs
were purified, resolved on a denaturing gel, analysed by northern
blot analysis with oligonucleotide probes specific to U1, U2, U4,
U5 and U6 snRNAs (Fig. 2A) and the efficiency of precipita-
tion was quantified (see legend of Fig. 2B). The eIF4G homo-
logues reproducibly pulled down U1 snRNA. More specifically,
~20% of the input levels of U1 snRNA were pulled down by
Tif4631p-TAP, whereas ~6% was precipitated by Tif4632-TAP
(Fig. 2A and B), while more than 60% of the input U1 snRNA
levels were precipitated under the same conditions by the TAP-
tagged yeast CBP20 homologue, Mud13p (data not shown). The
interaction of Tif4631p with U1 snRNA was reduced to ~10% of
the input levels in increased salt concentration, whereas the less
efficient interaction of this particular UsnRNA with Tif4632p
could withstand salt much better, remaining at similar levels at
350 mM NaCl (Fig. 2A and B). Furthermore, ~7% of the input
levels of U6 snRNA and ~10% of U4 snRNA were also pulled
down by both eIF4G homologues, however these amounts were
significantly reduced by increased salt concentration, mainly for
Tif4631p and to a lesser extent for Tif4632p (Fig. 2B). Finally,
surveillance mechanism comprising the recognition and subse-
quent degradation of mRNAs bearing a premature termination
codon.21 Research in human cell lines suggests that both nuclear
and cytoplasmic NMD occur on CBC-associated rather than
eIF4E-associated mRNA, suggesting a role for CBC in transla-
tion.22 ,23 In mammalian cells, NMD is translation and splicing
dependent.24-27 Ferraiulo et al.28 reported that translation initia-
tion factor eIF4AIII, a mammalian nucleo-cytoplasmic shuttling
protein that interacts physically or functionally with eIF4G, is
loaded onto the mRNA during splicing in the nucleus and then
functions during NMD, indicating one more link between
nuclear and cytoplasmic RNA processing events.
In Saccharomyces cerevisiae the domain of eIF4G responsible
for the interaction with CBC resides between the eIF4E binding
motif and the MIF4G domain.29 Fortes et al.29 proposed a role for
the CBC-eIF4G interaction in the exchange of CBC for eIF4E
and/or the direct recruitment of nascent mRNA for translation.
However, it is not known whether this interaction occurs in the
cytoplasm or in the nucleus. The latter can not be excluded since
CBC as well as many components of the translation machinery
are present in both cellular compartments.4,3 0-32 In S. cerevisiae
the interaction between eIF4G and CBC is not required for the
first round of mRNA translation that proceeds NMD, while the
exact role of this interaction is yet to be defined.33 Yeast two-
hybrid experiments have previously suggested that S. cerevi-
siae splicing factors, such as Prp11p and Snu71p could interact
with the yeast eIF4G protein,34 and the interaction of Prp11p
with Tif4631p was proposed by Ho et al.35 in a high through-
put mass spectrometric protein complex identification screen.
In human cells, association of eIF4G with pre-mRNA and the
spliceosome, as well as partial co-localization of nuclear eIF4G
Figure 1. Yeast eIF4G homologues are distributed between the
nucleus and the cytoplasm. Wild-type yeast cells (control) and cells
expressing TIF4631:TAP or TIF4632:TAP were grown to OD600: 0.2–0.4
and immobilized on slides as described in Materials and Methods. The
location of Tif4631p and Tif4632p was detected with rabbit IgG Alexa
488 labeled (B). DAPI staining was used for the localization of the nuclei
(C). The merged image is also presented in (A).
www.landesbioscience.com RNA Biology 565
a specific association between Prp11p and Tif4631p, character-
ized by the presence of two pools of complexes, since a great per-
centage of the interaction was lost at increased salt concentration
less than 5% of the input levels of U5
and U2 snRNAs were precipitated
by the eIF4G homologues, and these
interactions were greatly abolished
when salt was increased to 350 mM
NaCl (Fig. 2B). There was no sig-
nificant precipitation of UsnRNAs
from whole cell extracts prepared
from the isogenic wild type strain
(Fig. 2A, lanes 2 and 3) indicating
that the detected RNAs were pulled
down via their interaction with
Tif4631-TAP and Tif4632-TAP pro-
teins. These results suggest a specific
interaction of both Tif4631p and
Tif4632p mainly with U1 snRNA,
with the interaction of Tif4631p and
U1snRNA being more prominent
(Fig. 2). In addition, both proteins
can pull down to a similar extent U4
and U6 snRNAs (Fig. 2B).
Interaction of Tif4631p and
Tif4632p with protein components
of U1 and U2 snRNPs. There is evi-
dence from yeast 2-hybrid screens
that Tif4631p can interact specifi-
cally with the U2 snRNP protein,
Prp11p and the U1 snRNP specific
protein Snu71p, whereas Tif4632p
was found to interact with Prp11p.34
To assess these interactions GST-
Prp11p and GST-Snu71p fusion pro-
teins were expressed in E. coli and
bound to Glutathione-Sepharose
beads. The immobilized proteins
were incubated with 35S-labeled
Tif4631p or Tif4632p and the puri-
fied complexes were analysed by
SDS-PAGE and fluorography. Both
Tif4631p and Tif4632p were pulled
down by the immobilized GST-
Prp11p very efficiently (Fig. 3A,
lanes 1 and 2). Both proteins were
also pulled down by GST-Snu71p,
although in substantially lower
amounts than with GST-Prp11p
(Fig. 3A, lanes 7 and 8), whereas no
protein was bound to Glutathione-
Sepharose beads alone (lanes 4–6),
indicating that the observed binding
was specific. Incubation of the immo-
bilized GST-Prp11p with 35S labelled
Snu71p and analysis of the purified
complexes gave no detectable signal (Fig. 3A, lane 3), showing
that the interaction detected between Tif4631p, Tif4632p and
GST-Prp11p and -Snu71p is specific. We propose that there is
Figure 2 . Yeast eIF4G homologues can precipitate U1 and U2 snRNA in vitro. (A) Yeast extracts
derived from strains expressing either Tif4631p-TAP (lanes 4 –6) or Tif4632p-TAP (lanes 7–9), as well
as the isogenic wild-type strain (lanes 1–3), were incubated with IgG-Sepharose beads under increas-
ing salt concentration (lanes 2 , 5, 8: 150 mM NaCl, lanes 3, 6, 9: 350 mM NaCl). The co-precipitated
RNAs, as well as 20% of the input RNAs (lanes 1, 4, 7), were assayed by northern blot ting, using specic
oligonucleotide-probes for each of the U1, U2, U4, U5 and U6 snRNAs. (B) Densitometry and Phospho-
rimager analysis were both used to quantify the signal produced by the northern analysis presented in
(A), using the TotaLab software (Nonlinear Dynamics, UK) and ImageQuant software respectively. The
results presented are means ± SEM from three independent experiments.
566 RNA Biology Volume 6 Issue 5
whereas deletions extending beyond amino
acid 567 resulted in loss of interaction with
GST-Snu71p (Fig. 4A). The above suggest
that the minimal region of Tif4631p required
for interaction with Prp11p resides between
residues 494 and 529 (Fig. 4A).
To define in further detail the minimal
domain of Tif4631p required for interac-
tion with Prp11p, 10aa deletion mutants of
Tif4631p were expressed and assayed in pull
down assays with GST-Prp11p as described
above. Figure 4B shows that individual dele-
tion of aa 491–501, 534–544 or 566–576
reduced significantly the amount of Tif4631p
mutant that could be pulled down by GST-
Prp11p (lanes 2–4, compared to lane 1),
suggesting that these residues contribute to
the interaction between these proteins. It is
noteworthy that none of these deletions was
enough to abolish the interaction completely.
In addition, substitution of residues 496–
498 or 506–508 with Ala residues showed
that these specific amino acids, and more
crucially residues 506–508 are important
for the interaction of Tif4631p with GST-
Prp11p (Fig. 4B, lanes 6, 7 compared to lane
1). On the other hand, deletion of residues
457–467, 469– 479 (Fig. 4C, lane 5 and data
not shown), as well as 565–647 (Fig. 4C)
did not affect significantly the levels of Tif4631p mutant that
could be pulled down by GST-Prp11p, as expected (Fig. 4A).
Taken together these findings suggest that the absolutely essen-
tial region of Tif4631p required for the interaction with Prp11p
resides between amino acids 508–529. It is obvious, however,
that all amino acids within the region of 494–529 contribute to
the high affinity interaction of Tif4631p with Prp11p, since dele-
tion of individual domains within this region, in the context of
an otherwise full-length protein do not abolish the interaction
completely. The fact that a Tif4631p mutant lacking aa 504 to
952 can not be pulled down by GST-Prp11p (Fig. 4C) and nei-
ther can be a mutant lacking residues 1–529 (Fig. 4A), verifies
that residues 504–529 are absolutely required for the interaction
of Tif4631p with GST-Prp11p in our pull down assays. Using the
same approach we mapped the region of Tif4631p interacting
with Snu71p between amino acids 567 and 647 (Fig. 4A) and
that was verified by the finding that deletion of residues 565–647
of Tif4631p resulted in loss of the interaction of this protein with
GST-Snu71p in our pull-down assays (Fig. 4D, lane 4).
Fortes et al.29 found that amino acids 490–592 are required
for the interaction of Tif4631p with yeast CBC. The fact that
this region includes also the residues necessary for interaction
with Prp11p and Snu71p, as described above, prompted us to
investigate whether we could characterize in more detail the
individual interactions of this domain. Using our Tif4631p trun-
cated and deletion mutants we found that deletion of amino acids
491–501 resulted in more than 30% reduction of the interaction
(up to 500 mM NaCl, Fig. 3B, lanes 1–3) while a small but
significant percentage was persistent even at very high salt con-
centration. The interaction of Tif4631p with Snu71p was less
resistant to the highest salt concentration used (Fig. 3B, lanes
6–8). RNase A treatment did not significantly alter the bind-
ing pattern, demonstrating that the interaction of Tif4631p and
Tif4632p with both Prp11p and Snu71p is not RNA mediated
(Fig. 3B, lanes 9–11, and data not shown).
Mapping of the domain of Tif4631p that interacts with
splicing factors. To map the domains of Tif4631p that are
responsible for the interactions with both Snu71p and Prp11p,
N-terminal and C-terminal truncated forms of Tif4631p were
generated, in vitro translated and used in GST pull down experi-
ments using immobilized GST-Prp11p or GST-Snu71p (Fig. 4A).
Deletion of residues 657–952 from the C-terminus of Tif4631p
had no effect on binding efficiency of this protein to either
GST-Prp11p or GST-Snu71p. Further deletion of amino acids
593–656 resulted in about 30% less efficient binding, whereas
complete loss of pull-down efficiency was evidenced when amino
acids 267–592 were also deleted (Fig. 4A). In addition, delet-
ing the N-terminal residues up to amino acid 452 did not affect
the binding efficiency of Tif4631p for either Prp11p or Snu71p
(Fig. 4A). Further deletion of amino acids 453–493 resulted in
approximately 50% reduction of the interaction efficiency with
Prp11p, while still maintaining the interaction with Snu71p
(Fig. 4A). After sequential deletion of amino acids 494–528 the
truncated Tif4631p could not be pulled down by GST-Prp11p,
Figure 3. Yeast eIF4G homologues interact with protein components of U1 and U2 snRNPs.
(A) Immobilized GST-Prp11p (lanes 1–3) or GST-Snu71p (lanes 7 and 8) were incubated with
35S-labeled Tif4631p, Tif4632p or Snu71p as described in Materials and Methods. The pulled-
down proteins were resolved by SDS-PAGE, and visualized by autoradiography. GST alone
immobilized on Glutathione- Sepharose beads was used as negative control (lanes 4–6). 1/20 of
input 35S -labeled proteins were also analysed (lanes 9–11). (B) Immobilized GST-Prp11p (lanes
1–3, 10 and 11) or GST-Snu71p (lanes 6–8) or GST alone (lanes 4 and 5) were incubated with
35S-labeled Tif4631p, in the presence of increasing amounts of NaCl, or in the presence (+) or
absence (-) of 200 ng/ml RNase A as indicated. The pulled-down proteins were resolved by
SDS-PAGE, and visualized by autoradiography. 1/10 of the input 35S -labeled proteins were also
analysed (lane 9).
www.landesbioscience.com RNA Biology 567
Figure 4. For gure legend, see page 568.
568 RNA Biology Volume 6 Issue 5
increasing amounts of these conserved domains fused to GST
at their N-terminus. Addition of either GST-Tif4631p(453-647)
(Fig. 5A, lanes 3–5) or GST-Tif4632p(424-609) (lanes 6–8)
resulted in inhibition of splicing, as judged by the production
of less intermediate (intron-3' exon) and final (intron-lariat)
products of splicing, compared to the control (Fig. 5A, lane 2).
Addition of GST alone had a minor effect in the efficiency of the
splicing reaction (Fig. 5A, lanes 9–11). Consequently, addition
of increasing amounts of either the Tif4631p or the Tif4632p
domains in the splicing reaction resulted in an increasing inhibi-
tory effe ct ( Fig . 5B ). Addition of the GST-Tif4631p mutant lack-
ing amino acids 534–544, and therefore unable to interact with
yeast CBP20, in in vitro splicing reaction showed that this domi-
nant negative effect could not be attributed to selective sequestra-
tion of CBC by Tif4631p (data not shown). The above findings
suggest that inhibition of splicing caused by Tif4631p(453-647)
and Tif4632p(424-609) is possibly due to sequestration of spli-
ceosomal components like Prp11p and Snu71p away from the
spliceosomal machinery.
Deletion of TIF4631 results in accumulation of certain pre-
mRNAs in vivo. The interaction of Tif4631p and Tif4632p with
components of U1 and U2 snRNPs, together with the ability of
Tif4631p(453-647) and Tif4632p(424-609) domains to inhibit
splicing in vitro, suggested a possible role for the yeast eIF4G
homologues in splicing. To test this hypothesis, given that double
deletion of the yeast eIF4G homologues is lethal,19 we assayed
splicing in vitro using extract prepared from a strain deleted
of TIF4631, and expressing TAP-tagged Tif4632p that could
sequentially be depleted by IgG-Sepharose beads. Analysis of the
RNA products of splicing showed no significant difference in the
levels of splicing between control and TIF4631 deleted/Tif4632p
depleted extracts, indicating that Tif4631p or Tif4632p could
not be made limiting for efficient splicing in vitro. Western blot
analysis performed to check the depletion level of Tif4632p in the
extracts showed that we could not completely deplete Tif4632p.
Consequently, we can not completely exclude the possibility that
Tif4631p and Tif4632p play a role in splicing, since we possi-
bly could not reduce yeast eIF4G homologues to limiting levels
for splicing in vitro. We therefore focused our attention on splic-
ing in vivo by assaying the splicing efficiency of different pre-
mRNAs globally in the wild type and tif4631-∆ strains using
efficiency with TAP-Mud13p (Fig. 4E, lanes 4 and 5) and we
identified the minimal region of Tif4631p required for interac-
tion with TAP-tagged yeast CBP20 (Mud13p) in vitro to reside
between residues 522 and 612 (data not shown). In addition,
we identified a Tif4631p deletion mutant lacking amino acids
534–544, that could still bind to GST-Prp11p, but had abolished
completely the interaction with TAP-Mud13p (Fig. 4E, lanes 3
and 6). This finding indicates that residues 534–544 of Tif4631p
are essential for the interaction with yeast CBC. More impor-
tantly, the fact that Tif4631p-∆(534-544) interacts with Prp11p
but not with Mud13p shows that Tif4631p can interact with
Prp11p in a CBC-independent manner. A summary of the above
described domains, as well as a schematic representation of the
domains of Tif4631p that are known to be required for interac-
tion with Pabp1p36 and eIF4E,37 as well as the MIF4G domain17
are shown for comparison in Figure 4E.
Sequence comparison shows that the region of Tif4631p that
interacts with Prp11p, Snu71p and Mud13p is also very highly
conserved in Tif4632p.19 Using the same method as for Tif4631p,
N-terminal and C-terminal truncation mutants of Tif4632p were
assayed for their efficiency to bind to Prp11p and Snu71p and
amino acids 424–609 were defined as the minimum region of
Tif4632p required for interaction with Prp11p and Snu71p (data
not shown).
Inhibition of splicing in vitro by Tif4631p/Tif4632p
interaction domains. As shown in Figure 3C, the binding of
Tif4631494-952 to Prp11p was noticeably less than that observed
with Tif4631453 -952 . To obtain the minimum domain of Tif4631p
that interacts efficiently with U1 and U2 snRNP proteins we
constructed a Tif4631p domain mutant spanning amino acids
453 to 647, so as to include the region of Tif4631p that interacts
with Snu71p. We refer to this construct as “Tif4631p(453-647)”.
Similarly, a construct with the Tif4632p domain required for
interaction with both Prp11p and Snu71p was made and we refer
to this construct as “Tif4632p(424-609)”. After verifying, by
pull-down experiments with GST-Prp11p and GST-Snu71 pro-
teins, that domains Tif4631p(453-647) and Tif4632p(424-609)
interact specifically with both Prp11p and Snu71p in vitro (data
not shown), we used these constructs to determine whether
Tif4631p(453-647) and Tif4632p(424-609) can influence
splicing by supplementing in vitro splicing reactions with
Figure 4. Detailed mapping of the interaction of Tif4631p with Snu71p and Prp11p. (A) Schematic representation of the deletion mutants of Tif4631p.
Pull-down experiments were performed by incubation of immobilized GST-Pp11p or GST-Snu71p with 35S-labeled full-length or truncated Tif4631p
mutants and analysed as described in Figure 3A. Densitometry was used to quantify the respective autoradiographs and the quantication results are
presented as % percentages of the input protein used for the pull-down experiments. (B) Immobilized GST-Prp11p (lanes 1–7) was incubated with
35S-labeled Tif4631p (wt) or the Tif4632p -mut ant proteins indicated, as described in Figure 3. The pulled-down proteins were resolved by SDS-PAGE,
and visualized by autoradiography. 1/5 of input 35S-labeled proteins were also analysed (lanes 8–15). (C) Immobilized GST-Prp11p (lanes 4–6) was
incubated with 35S-labeled Tif4631p (wt) or the Tif4632p mutants indicated. The pulled- down proteins were resolved by S DS-PAGE, and visualized by
autoradiography. GST alone immobilized on Glutathione- Sepharose beads was used as negative control (lanes 7–9). 1/5 of input 35S-labeled proteins
were also analysed (lanes 1–3). The full length 35S-labeled proteins are indicated by arrows. (D) Immobilized GST-Snu71p (lanes 4 – 6) were incubated
with 35S -labeled Tif4631p (wt) or the Tif4632p mutants indicated. The pulled-down proteins were resolved by SDS-PAGE, and visualized by autoradiog-
raphy. GST alone immobilized on Glutathione-Sepharose beads was used as negative control (lanes 7–9). 1/5 of input 35S-labeled proteins were also
analysed (lanes 1–3). The full length 35S-labeled proteins are indicated by arrows. (E) Immobilized GST-Prp11p (lanes 1–3) or TAP-Mud13p (lanes 4 – 6)
were incubated with 35S-labeled wild-type Tif4631p (wt) or Tif4631p mutants lacking amino acid residues 491–501 (∆491-501), or 534– 544 (∆534-54 4).
The pulled-down proteins were resolved by SDS -PAGE and visualized by autoradiography. 1/5 of the input 35S-labeled proteins was also analysed for
comparison. (F) Schematic represent ation of the domain of Tif4631p required for interaction with Prp11p, Snu71p and CBC. Other characterized
domains of Tif4631p are also indicated (see text for details).
www.landesbioscience.com RNA Biology 569
promotes association of U1 snRNA with the cap proximal
5' splice site.2,12
Consistent with these results, we provide evidence here for
the interaction of yeast eIF4G homologues with spliceosomal
UsnRNPs. We show that mainly Tif4631p, and Tif4632p to
a lesser extent, interact with U1 snRNA. In addition, both
eIF4G homologues are also shown to interact stably with the
U1 snRNP component Snu71p, either directly or via other
proteins present in the reticulocyte lysate used in our assays.
This particular interaction, however, is much less efficient pos-
sibly because Snu71p alone does not mediate the interaction
of eIF4G proteins with U1 snR NP. Using pull-down binding
assays, we show that both eIF4G homologues interact very effi-
ciently and stably with Prp11p, a U2 snRNP component in an
RNA-independent manner, whereas our TAP-tagged eIF4G
homologues can only pull down a minor amount of U2 snR NA
in vitro. This finding suggests that the high affinity interaction
of yeast eIF4G proteins with Prp11p is independent of its inter-
action with U2 snRNA. The finding that eIF4G homologues
can pull down U4 and U6 snR NAs to a similar extent is not
unexpected since the existence of a penta-snRNP complex has
been proposed for yeast.41
splicing microarrays.38 Total RNA was extracted from the above
strains and hybridized to slides containing an array of oligonu-
cleotides able to hybridize to all the intron-containing RNAs of
S. cerevisiae and to distinguish between pre-mRNAs and
mRNAs.38 These oligonucleotides were complementary to the
5' exon-intron junction of each one of the S. cerevisiae pre-
mRNAs, or to the intron itself or to the exon-exon junction of
each one of the mature mRNAs (Fig. 6A). For data normaliza-
tion purposes oligonucleotides able to hybridize to the 3' exon of
each one of all the pre-mRNAs and mRNAs were also used.
Out of 257 genes tested, there were 6 that consistently, in five
independent experiments, showed accumulation of pre-mRNA
at least two-fold or more in the tif4631-∆ strain compared to the
isogenic control strain (Table 2). Similar analysis performed for a
tif4632-∆ strain did not show any significant change in the pre-
mRNA or mRNA levels, compared to the isogenic control strain.
The microarray results were then verified for the gene YJR145C,
for which accumulation of pre-mRNA could be detected in the
tif4631-∆ strain compared to the wt strain, using primer exten-
sion experiments (Fig. 6B, lanes 1 and 2; quantified in C). As
a positive control we used the prp2-1 strain, that is deficient in
splicing and exhibits a much stronger splicing block at the non-
permissive temperature (37°C)14 (Figs. 6B, lanes 3 and 4 and
5C). YJR145C is the systematic name used for the gene RPS4A,
which gives rise to the ribosomal protein Rps4Ap, a protein
identical to Rps4Bp. The primer used for the primer extension
experiment could hybridize to pre-mRNA and mRNA entities
representing both genes. A two- to three-fold accumulation of
the pre-mRNAs corresponding to both RPS4A and RPS4B could
be detected in the tif46321-∆ strain compared to the isogenic
control strain, whereas strain prp2-1 showed the expected strong
splicing deficient phenotype (Fig. 6C).
Discussion
In the present study, we investigate the putative role of the yeast
translation initiation factor eIF4G in nuclear pre-mRNA pro-
cessing. Using in situ localization we show that, in addition to
their expected cytoplasmic localization, eIF4G homologues of
S. cerevisiae, Tif4631p and to a smaller extent Tif4632p, exhibit
a significant presence in the yeast nucleus. This agrees with pre-
vious reports that have demonstrated the presence of eIF4G in
the nucleus of HeLa cells, indicative of a possible role for eIF4G
in nuclear RNA processing.20,39 Huh et al.
40 however, report
that both yeast eIF4G homologues are located in the cytoplasm,
using C-terminally GFP-tagged fusion constructs, fluorescent
microscopy and an experimental approach different to ours.
In the cytoplasm, eIF4G plays an essential role in translation
by acting as an adapter molecule during the initiation phase
of protein synthesis. Within its sequence it contains domains
that interact with eIF4E, eIF4A, eIF3, PABP and Mnk1.14 In
addition, S. cerevisiae eIF4G has been shown to interact with
the cap binding complex, CBC, via a domain between eIF4E
and eIF3 binding sites.29 McKendrick et al.20 have shown that,
in mammalian cells, a nuclear pool of eIF4G is closely associ-
ated with CBC. Moreover, it is known that CBC in the nucleus
Figure 5. Tif4631p(453-647) and Tif4632p(424-609) inhibit splicing in
vitro. (A) Increasing amounts (0.2–1 μg) of a GST-Tif4631p(453-647)
(lanes 3–5) or GST-Tif4632p(424-609) (lanes 6– 8) or GST alone (lanes
9–11), were added to in vitro splicing reactions, and the RNA products
were analyzed by denaturing gel electrophoresis and autoradiography.
Lane 1 shows the pre-mRNA used and lane 2 shows the control reac-
tion without the addition of any protein. (B) Histogram derived from
the quantication of data presented in (A) by phosphorimager analysis.
Both spliced products (lariat and lariat exon) were quantied and are
represented as a percentage of the unspliced pre-mRNA .
570 RNA Biology Volume 6 Issue 5
sequestering them away from the
splicing machinery, supporting the
hypothesis that eIF4G proteins can
participate in pre-mRNA processing
events.
To further investigate the possible
role of yeast eIF4G in splicing, we
undertook a global analysis of pre-
mRNA splicing using microarrays.
A subset of six pre-mRNAs showed a
reproducible two- to three-fold accu-
mulation of pre-mRNAs in strains
deleted of the TIF4631 compared to
the isogenic control (Table 2). Even
though the possibility of this being an
indirect effect can not be ruled out,
it is interesting that the same result
was not observed when this microar-
ray analysis was applied on a strain
deleted of the TIF4632, where no
accumulation of any pre-mRNA was observed. Our results show
that predominantly Tif4631p interacts with spliceosomal U1
snRNP in vitro, and this interaction could be possibly required
for efficient processing of certain pre-mRNAs. Analysis and com-
parison of intron sequences and splice sites of the pre-mRNAs
presented in Table 2 did not reveal any obvious common features
between these RNAs, apart from the fact that they are all pre-
mRNAs that will give rise to ribosomal proteins. In addition,
Fine mapping of the individual domains of eIF4G proteins
required for the interaction with Prp11p, CBC and Snu71p shows
that they are distinct, but adjacent to each other and together
they form an “interaction domain” that resides in a region that
is highly conserved between the two yeast eIF4G isoforms.
Addition of a recombinant form of the above defined “interac-
tion domain” of yeast eIF4G can efficiently inhibit splicing in
vitro, possibly by interacting with spliceosomal components and
Figure 6. Accumulation of pre-
mRNAs in tif 4631-∆ strain in vivo. (A)
Schematic overview of the splicing
microarray. Four dif ferent probes able
to detect intermediate species of the
two stages of splicing were designed
and used on the microarray. The intron
probe (black), as well as the 5'-exon-
intron probe (dark grey) can only be
hybridized to by un-spliced RNA. The
mature junction probe (grey) will only
be hybridized to by processed mRNA,
whereas the exon 2 probe (white) will
be hybridized to by both pre-mRNA
and mRNA and is used as a control to
remove effects due to dif ferential gene
expression. (B) Total RNA was ex-
tracted from the ti f46 31-∆ strain as well
as from the isogenic wild-type and was
used in primer extension experiment,
as described in Materials and Methods.
The cDNAs were analysed by denatur-
ing gel electrophoresis and autoradiog-
raphy. RNA from strain prp2-1, grown
either at 23°C or at 37°C was also used
in the same experiment as a control.
The positions of both the mature and
precursor mRNAs for both RPS4A and
RPS4B are indicated. (C) Quantication
of the bands corresponding to both
pre-mRNAs and mRNAs of the gel
presented in (B) by phosphorimaging.
www.landesbioscience.com RNA Biology 571
5'-GGA TCC TAA TAC GAC TCA CTA TAG GAA CAG
ACC ACC ATG ACA CCA ATT GAA GAT GTC-3' for amino
acids 422–952, 5'-GGA TCC TAA TAC GAC TCA CTA TAG
GAA CAG ACC ACC ATG GGT CCT GAT ATC AAA TAC for
amino acids 438–952, 5'-GGA TCC TA A TAC GAC TCA CTA
TAG GAA CAG ACC ACC ATG CCA ACT TTC TTG CTT
C-3' for amino acids 453–952, 5'-GGA TCC TAA TAC GAC
TCA CTA TAG GA A CAG ACC ACC ATG GGA GAT TCT
GGC AGA TTC GGC-3' for amino acids 494–952, 5'-GGA
TCC TAA TAC GAC TCA CTA TAG GA A CAG ACC ACC
ATG AGA AGA TCA AAG AGA-3' for amino acids 529–952,
5'-GGA TCC TAA TAC GAC TCA CTA TAG GAA CAG ACC
ACC ATG AAG GA A GAA GTT GCT CC-3' for amino acids
567–952, 5'-GGA TCC TAA TAC GAC TCA CTA TAG GAA
CAG ACC ACC ATG GAC GGA AAG ACC GAC TAT TGG-
3' for amino acids 596–952, 5'-GGA TCC TAA TAC GAC TCA
CTA TAG GA A CAG ACC ACC ATG GCT ATT GCA AAC
ATA TCA G-3' for amino acids 631–952, 5'-GGA TCC TAA
TAC GAC TCA CTA TAG GA A CAG ACC ACC ATG GCT
GTG ATA GAA CAG-3' for amino acids 647–952. The PCR
products were purified and used directly in in vitro transcription/
translation reactions with rabbit reticulocyte lysates according to
manufacturer’s instructions (Promega) to obtain the 35S-labeled
truncated versions of Tif4631p.
To construct deletion mutant Tif4631p-∆(565-647), the PCR
product produced by forward primer 5'-TAT ACA TAT GGC
CCA ACT TTC-3' and reverse primer 5'-CGA CGA TAT CGT
CCT CTC TTT TC-3' on pBS-Tif4631 as a template, was
digested with NdeI/EcoRV and the PCR product produced by
forward primer 5'-CAC CGA TAT CGA ACA GAT TTT C-3'
and reverse primer 5'-CGC AGG ATC CAA GAG AAT GAA
TGA C-3' was digested with EcoRV/SacII. The digested PCR
products were ligated with each other and the pBS-Tif4631 frag-
ment derived from NdeI/SacII digest. For construction of pBS-
Ti f4631-∆(504-952), pBS-Tif4631 was digested with MscI/StuI
and the respective fragment was gel extracted and ligated.
The strains of S. cerevisiae used in this study are described in
Table 1. Standard yeast growth conditions and manipulations
were used.
In situ localization. Yeast strains TIF4631::TAP or
TIF463::TAP were grown to OD600 = 0.2–0.4 and paraformal-
dehyde (EMS) was added to final concentration 4%. Cells were
harvested, washed in buffer B (1.2 M sorbitol, 65 mM KH2PO4,
35 mM K2HPO4) at 4°C and resuspended in 1 ml of buffer B.
five of these R NAs are characterized by the presence of a non-
consensus 5' splice site. The interaction of eIF4G with UsnRNPs
during splicing in vivo could be possibly necessary as a check
point during the processing of these pre-mRNAs. In support
of this, a partial but significant co-localization of eIF4G with a
proportion of snRNPs at discrete foci was reported in mamma-
lian cells,20 a finding that strengthens the hypothesis that eIF4G
might participate in the processing of some pre-mRNAs in the
nucleus.
In conclusion, we present here a detailed mapping of the
domains of yeast eIF4G required for interaction with compo-
nents of the spliceosomal machinery. These interactions probably
implicate eIF4G in nuclear RNA processing events in S. cerevisiae.
Even though yeast eIF4G interacts specifically with spliceosomal
components, it is not required for splicing of the RNAs tested in
vitro, but a small but significant accumulation of pre-mRNAs is
observed in a tif46 31- ∆ strain in vivo. A tif4632-∆ strain does not
exhibit the same effect, a finding that might explain the normal
growth phenotype of this strain, in contrast to the slow growth
phenotype of tif 4631-∆ strain reported by Goyer et al.19 At present
we can not explain why the genetic depletion of one of the eIF4G
homologues in yeast affects the splicing of a small number of pre-
mRNAs that correspond to ribosomal proteins. There is evidence
of an auto-regulatory role for alternative splicing coupled NMD
in the expression of a number of ribosomal proteins,42-44 while
MIF4G,17 a domain that is conserved between eIF4G, Upf2p/
NMD2p and CBP80 implicates eIF4G and CBP80 in nonsense-
mediated decay. One could suggest that eIF4G participates in
nuclear RNA processing by linking in some way pre-mRNA
splicing with degradation pathways like NMD and acting once
more as a scaffold protein that brings important protein compo-
nents together.
Materials and Methods
Plasmids and S. cerevisiae strains. Plasmids pBS-Tif4631 and
pBS-Tif4632 were used as templates for coupled in vitro transcrip-
tion-translation reactions in rabbit reticulocyte lysates to express
[35S]-labeled eIF4G proteins. They were constructed by sub-
cloning PCR amplified TIF46 31 and TIF4632 genomic DNAs
respectively into pBS(SK-) (Stratagene) as EcoR1/BamH1 frag-
ments with primers 5'-GGG AAT TCA TGA CAG ACG A AA
CTG TCA AC-3' and 5'-CCG GAT CCT TAC TCT TCG TCA
TCA CTT TCT-3' for TIF46 31 and 5'-GGG AAT TCA TGA
CTG ACC AAA GAG GTC CAC-3' and 5'-CCG GAT CCT
TAA TCA CTG TCC CCA TCG TTA-3' for TIF4632. Plasmid
pGEX4T-1-Prp11 was used to express GST-Prp11p. The ORF of
PRP11 was subcloned as a Sal1/SmaI fragment from pAS2DD-
pr p11,34 into pGEX4T-1 XhoI/SmaI restriction sites. To produce
plasmids expressing C-terminally truncated versions of Tif4631,
pBS-TI F46 31 was d ige ste d with ea ch of B stBI, S tuI, NheI a nd B lpI
to obtain the 1–765, 1–656, 1–592 and 1–266 truncated forms of
Tif4631p respectively. To obtain N-terminally truncated versions,
a reverse primer 5'-CTT ACT CTT CGT CAT CAC TTT CTC
CC-3' and the following forward primers were used to amplify by
PCR the fragments of TIF4631 referred next to each primer:
Table 1. S. cerevisiae str ains used in this study
Name Genotype
TIF4631::TAP MATa ade2-101 his3-∆200 leu2-∆1 trp1-∆99 ura3-∆ 99
cir0tif4631-TAP::TRP1
TIF4632::TAP MATa ade2-101 his3- ∆200 leu2-∆1 trp1-∆99 ura3-∆ 99
cir0tif4632-TAP::TRP1
ti f46 31- ∆MATalpha ade2 his3 leu2 trp1 ura3(GAL+) tif4631∆::HIS3
tif4632- ∆MATalpha ade2 his3 leu2 trp1 ura3(GAL+) tif4632∆::HIS3
ti f46 31- ∆;
TIF4632::TAP
MATa ade2-101 his3- ∆200 leu2-∆1 trp1-∆99 ura3-∆ 99
cir0tif4632-TAP::TRP1 tif4631∆::HIS3
572 RNA Biology Volume 6 Issue 5
OD600 = 2 and cells were harvested and lysed in buffer A (10 mM
HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT,
0.5 mM PMSF, 1 μM leupeptin, 2 μM pepstatin A, 4 μM chy-
mostatin, 2.6 μM aprotinin) by French press. The TAP-tagged
proteins were purified by incubating the yeast cell lysates with 50
μl of a 50% solution of IgG Sepharose beads for 2.5 hrs at 4°C.
To analyse the RNAs that are pulled down by Tif4631-TAP or
Tif4632-TAP proteins, the beads were washed extensively with
either IPP150 buffer (150 mM NaCl, 0.1% Nonidet P-40, 10 mM
Tris, pH 8.0) or IPP350 buffer (350 mM NaCl, 0.1% Nonidet
P-40, 10 mM Tris, pH 8.0) and then the RNA was extracted
by phenol/chloroform, precipitated and analysed on a 6% acryl-
amide gel. Northern hybridization followed using the following
specific oligonucleotide probes: 5'-CTT AAG GTA AGT AT-3'
for U1, 5'-CTA CAC TTG ATC TAA GCC AA A AGG C-3' for
U2, 5'-A AT ATG GCA AGC CC-3' for U5, 5'-CTC TTT GTA
AA A CGG TTC-3' for U6 and 5'-CCG TGC ATA AGG AT-3'
for U4snRNA.
Preparation of whole cell extracts of yeast. The protocol
of Umen and Guthrie45 was used with minor modifications.
Namely, the yeast strain of interest was grown to OD600 = 0.5–1
at 30°C in 1L YPD medium. Cells were harvested by centrifuga-
tion at 5,000 rpm for 5 min (Beckman JLA10.500 rotor) and
washed twice with 20 ml of AGK buffer (10 mM HEPES pH
7.9, 1.5 mM MgCl2, 200 mM KCl, 10% Glycerol, 2 mM DTT).
Cells were resuspended in 0.4 cell pellet volume of AGK buffer
and squirted through a syringe into liquid nitrogen. The frozen
cell pellet was ground to a fine powder by mortar and pestle and
allowed to thaw. The thawed powder was centrifuged at 17,000
rpm for 30 min at 2°C (rotor JA25.50 Beckman). The superna-
tant was centrifuged at 40,000 rpm for 1 hr at 4°C (rotor 70.1Ti,
Beckman) and subsequently dialysed for 3 hrs against buffer D
(20 mM HEPES pH 7.0, 50 mM KCl, 0.2 mM EDTA, 20%
(v/v) glycerol) at 4°C, centrifuged at 13,000 rpm for 10 min and
frozen at -80°C.
In vitro splicing. In vitro splicing reactions were carried out
and analysed as described by Lin et al.46 Actin pre-mRNA was
transcribed from linearized by BamHI plasmid p283,47 which
contains an AluI fragment of the yeast actin gene cloned at a
SmaI site of pGEM vector.
Microarrays. Total RNA was prepared as described by
Schmitt et al.48 while the microarrays used were similar to those
described by Clark et al.49 More precisely, there were at least four
After incubation with 1 mg oxalyticase for 10 min at 30°C, cells
were washed with ice-cold buffer B and resuspended in 0.65 ml
of the same buffer. A 0.1 ml aliquot was put on to a coverslip,
allowed to stand for 30 min and then washed with 3 ml of buffer
B, replaced with 5 ml of methanol and left for 5 min at -20°C.
After a final wash with buffer B, cells were permeabilized by buffer
C (0.1% Triton X-100, 20 mM HEPES pH 7.9, 200 mM NaCl)
for 30 min. Primary antibody (Rabbit IgG Alexa 488 labelled,
Molecular Probes) was used in 1:100 dilution in buffer C, for 1
hr. Washes with buffer C followed and after briefly rinsing with
PBS the coverslip was mounted with vectashield with DAPI and
examined using a Leica confocal microscope with three lasers
giving excitation lines at 380, 488 and 543 nm. The data from
the channels were collected separately using narrow-bandpass
filter settings. In multiple staining experiments, the laser intensi-
ties and data collection settings were adjusted to avoid overlap
(bleedthrough) between channels. The coupled microscope was
a Leica DMIBRE equipped with a 633 water immersion objective
lens (numerical aperture, 1.4). Data sets were processed using
the Leica TCS NT, version 1.4.338, software package and were
subsequently exported into Adobe Photoshop version 5.5, and
Deneba Canvas version 7.02.
In vitro pull down. For purification of GST fusion proteins,
Escherichia coli extracts from BL21-codon plus cells expressing
either GST-Prp11p or GST-Snu71p fusion proteins were prepared
by inducing 1 liter culture of exponentially growing cells at OD600
of 0.6–0.8, for 3.5 hrs with 1 mM IPTG. Cells were harvested,
resuspended in PBS with 0.5% Triton X-100 and lysed by French
press. The lysate was incubated with 500 μl of Glutathione-
Sepharose beads for 2 hrs at 4°C. The beads were washed exten-
sively with PBS-0.5% Triton X-100 and used subsequently in
the binding assays. For in vitro transcription/translation reac-
tions of TIF46 31 or TIF4632, the TNT® system (Promega) and
plasmids pBS-TIF4631 or pBS-TIF4632 were used according to
manufacturer’s instructions. For binding assays, GST-fusion pro-
teins bound to glutathione-sepharose beads were incubated with
Tif4631p or Tif4632p produced with the TNT® system and the
reaction mixtures were incubated for 2 hrs at 4°C. After extensive
washing with PBS-0.5% Triton X-10 0, the proteins were eluted by
SDS sample buffer and analysed by SDS/PAGE. For the detection
of 35S proteins, the EA starter kit was used (Thistle Scientific).
Purification of Tif4631p-TAP, Tif4632p-TAP proteins.
Yeast strains TIF4631::TAP and TIF4632::TAP were grown to
Table 2. Genes showing a splicing deficiency in the yeast strain tif 4631-∆, their respective ORFs and the ratio of pre-mRNA in the tif4631-∆ strain
compared to the wt strain
Gene ORF tif4631-∆/wt Exon 1 5' splice site Branch point 3' splice site Intron size
YBR181C RPS6B 2.06 gaag guaugua uuuacuaacaa guauuauuuauaacag 352
YOR096W RP S7A 2.07 agaa guauguu cuuacuaacat uuuccuucuuuuauag 401
YGL18 9C RPS26A 2.22 agua guauguu guuacuaacua augaauauuuaauuag 368
YLR448W RPL6B 2 .33 acaa guaugug uauacuaacua gauaugucaauuauag 384
YHR203C RPS4B 2.89 gacc guauguu uuuacuaacaa acgauuuuucauauag 269
YJ R145 C RPS4A 3 .16 gacc guauguu uuuacuaacga auuuuuuuccguacag 256
Sequences of the 5' splice site, the branch point and the 3' splice sites, as well as the size of the corresponding introns are shown (according to Lopez
et al.51).
www.landesbioscience.com RNA Biology 573
10–16 hrs in a humid chamber. Then the arrays were washed in
1x SSC, 0.2% SDS followed by 0.1x SSC, 0.2% SDS and finally
0.1x SSC, all at room temperature for 5 minutes. Scanning was
performed in an Affymetrix 418 scanner and spot quantitation
was carried out by ImageQuant v3.1.
To normalize, the background corrected intensity value for
each spot was divided by the background corrected intensity value
from the corresponding 3'exon probe. More precisely, in a mutant
strain where the pre-mRNA of a specific gene is more than the
respective mRNA, one would see more RNA hybridized to the
oligonucleotides that complement the 5'exon-intron junction or/
and intron rather than the one complementary to the exon-exon
junction, when compared to the wt strain. The above signals
were compared to each other after normalization by the signal
the specific RNA gives with the oligonucleotide that hybridizes
to each 3' exon and recognizes it in both pre- and mature forms.
Additionally, the signal that this RNA gives on the position of
the 3'exon recognizing oligonucleotide probe should be equal to
the sum of the signal it gives with the 5'exon-intron recognizing
oligonucleotide probe, plus the signal from the hybridization with
the exon-exon junction recognizing oligonucleotide probe. On
the other hand, the same amount of RNA is expected to hybrid-
ize to the oligonucleotide probe that complements the 3' exon
of this RNA for both control and mutant strain, since the total
amount of the specific RNA should be equal in both strains.
For the primer extension experiments performed to check
the microarray results, total RNA prepared as described above
was hybridized with a 32P-labeled oligonucleotide probe with the
sequence 5'-AGA AAG ACA ATC AAT GGC A AG GA-3' that
hybridizes in exon 2 of the YJR145C pre-mRNA. The reverse
transcription reaction was performed as described by Bousquet-
Antonelli et al.50
Acknowledgements
We thank Heike Roesner for help with the mapping of eIF4G
interaction domains. We thank Skarlatos G. Dedos, and Tuija
A.A. Pöyry for critical reading and comments on the manuscript.
We also thank Prof. Richard J. Jackson (Dept. of Biochemistry,
University of Cambridge) for his support. Research in the labo-
ratory of J.L. was initially supported by the Medical Research
Council. E.T. and A.J. were funded by the RNOMICS project
QLG2-CT-2001-01554. J.D.B. was funded by the Wellcome
Trust grant 067311 to Prof. Jean Beggs. P.K. held a Marie-Curie
Fellowship (R81517).
oligonucleotide probes per S. cerevisiae transcript, containing an
intron, immobilized on to the microarray. One probe was com-
plementary to the 5'exon-intron junction of all the pre-mRNAs,
one probe was complementary to sequences of the intron, another
probe was complementary to the exon-exon junction of all the
mature mRNAs and finally there was also a probe that would
hybridize to the 3' exon of all the pre-mRNAs and mRNAs used
for normalization. For transcripts containing more than one
intron and/or different predictions of intron start and end points,
the 5' and mature exon-exon junction probes (and occasionally
the intron probe) were duplicated as necessary to identify all pos-
sible introns. These probes were synthesized by MWG-Biotech
AG and have an average length of 41.2 nucleotides. Ideally a probe
should have a Tm of 80–90°C (nearest neighbor algorithm),
where constraints of the target sequence would allow. Similarly
all the probes to a transcript were designed to have a Tm within
±2°C of each other where possible. These probes were printed in
triplicate on to poly-lysine slides (Sigma) in 3x SSC buffer at the
GTI (Chancellor’s Building, Royal Infirmary, Edinburgh) using
a GMS 417 (Affymetrix) printer. This microarray was hybridized
with Cy dye labeled cDNA derived from total RNA from the
mutant strain of interest and an isogenic wild type. The reverse
transcription reaction was performed in the presence of Cy dye
modified dUTP (Amersham). Either Cy3 or Cy5 was used to
label the mutant and the other to label the wild type cDNA,
including at least one occasion where the dyes were swapped from
their normal total RNA type (dye flip labeling). Thermoscript
(Invitrogen) was used to perform the reverse transcription reac-
tion at 47°C using the manufacturer’s supplied buffers and
20–50 μg of total RNA. Priming was from gene specific primers
designed to bind downstream to the 3' exon probe (45 μM total
concentration). The RNA was removed using 55 mM NaOH, 28
mM EDTA pH 8 and 65°C for 30 minutes before neutralization
with 120 mM Tris pH 7.5 at 4°C. The labeled cDNAs (mutant
and wild type), were combined and spun in YM-100 columns
(Millipore), using the manufacturer’s instructions, in 10 mM
Tris pH 7.5 (used to wash twice more) to filter-purify and con-
centrate. The arrays were blocked with 1-methyl-2-pyrrolidinone
(93% v/v) and succinic anhydride (1.64% w/v) in 68.5 mM boric
acid (pH 7 with NaOH) at room temperature for 20 minutes
and then pre-hybridized by 5x SSC, 0.1% (w/v) SDS, 1% (w/v)
BSA at 47°C for 45 minutes before hybridization. The hybridiza-
tion solution was 5x SSC, 0.4% (w/v) SDS and 20 μg of human
COT-1 DNA in 16 μl under a 22 x 22 mm coverslip at 47°C for
References
1. Izaurralde E, Lewis J, McGuigan C, Jankowska M,
Darzynkiewicz E, Mattaj IW. A nuclear cap binding
protein complex involved in pre-mRNA splicing. Cell
1994; 78:657-68.
2. Lewis JD, Gorlich D, Mattaj IW. A yeast cap binding
protein complex (yCBC) acts at an early step in pre-
mRNA splicing. Nucleic Acids Res 1996; 24:3332-6.
3. Flaherty SM, Fortes P, Izaurralde E, Mattaj IW,
Gilmartin GM. Participation of the nuclear cap bind-
ing complex in pre-mRNA 3' processing. Proc Natl
Acad Sci USA 1997; 94:11893-8.
4. Gorlich D, Kraft R, Kostka S, Vogel F, Hartmann E,
Laskey RA, et al. Importin provides a link between
nuclear protein import and U snRNA export. Cell
1996; 87:21-32.
5. Visa N, Izaurralde E, Ferreira J, Daneholt B, Mattaj
IW. A nuclear cap-binding complex binds Balbiani ring
pre-mRNA cotranscriptionally and accompanies the
ribonucleoprotein particle during nuclear export. J Cell
Biol 1996; 133:5-14.
6. Shen EC, Stage-Zimmermann T, Chui P, Silver PA.
The yeast mRNA-binding protein Npl3p interacts
with the cap-binding complex. J Biol Chem 2000;
275:23718-24.
7. Furuichi Y, LaFiandra A, Shatkin AJ. 5'-Terminal struc-
ture and mRNA stability. Nature 1977; 266:235-9.
8. Shatkin AJ. mRNA cap binding proteins: Essential fac-
tors for initiating translation. Cell 1985; 40:223-4.
9. Ohno M, Kataoka N, Shimura Y. A nuclear cap bind-
ing protein from HeLa cells. Nucleic Acids Res 1990;
18:6989-95.
10. Colot HV, Stutz F, Rosbash M. The yeast splicing fac-
tor Mud13p is a commitment complex component and
corresponds to CBP20, the small subunit of the nuclear
cap-binding complex. Genes and Dev 1996; 10:1699-
708.
11. Mazza C, Segref A, Mattaj IW, Cusack S. Large-scale
induced fit recognition of an m(7)GpppG cap analogue
by the human nuclear cap-binding complex. EMBO J
2002; 21:5548-57.
12. Lewis JD, Izaurralde E, Jarmolowski A, McGuigan C,
Mattaj IW. A nuclear cap-binding complex facilitates
association of U1 snRNP with the cap-proximal 5'
splice site. Genes Dev 1996; 10:1683-98.
574 RNA Biology Volume 6 Issue 5
39. Etchison D, Etchison JR. Monoclonal antibody-aided
characterization of cellular p220 in uninfected and
poliovirus-infected HeLa cells: subcellular distribu-
tion and identification of conformers. J Virol 1987;
61:2702-10.
40. Huh W-K, Falvo JV, Gerke LC, Carroll AS, Howson
RW, Weissman JS, O’Shea EK. Global analysis of
protein localization in budding yeast. Nature 2003;
425:686-91.
41. Stevens SW, Ryan DE, Ge HY, Moore RE, Young
MK, Lee TD, Abelson J. Composition and functional
characterization of the yeast spliceosomal penta-snRNP.
Mol Cell 2002; 9:31-44.
42. Cuccurese M, Russo G, Russo A, Pietropaolo C.
Alternative splicing and nonsense-mediated mRNA
decay regulate mammalian ribosomal gene expression.
Nucleic Acids Res 2005; 33:5965-77.
43. Dabeva MD, Warner JR. Ribosomal protein L32 of
Saccharomyces cerevisiae regulates both splicing and
translation of its own transcript. J Biol Chem 1993;
268:19669-74.
44. Mitrovich QM, Anderson P. Unproductively spliced
ribosomal protein mRNAs are natural targets of mRNA
surveillance in C. elegans. Genes Dev 2000; 14:2173-
84.
45. Umen JG, Guthrie C. Mutagenesis of the yeast gene
PRP8 reveals domains governing the specificity and
fidelity of 3' splice site selection. Genetics 1996;
143:723-39.
46. Lin R-J, Newman AJ, Cheng S-C, Abelson J. Yeast
mRNA splicing in vitro. J Biol Chem 1985; 260:14780-
92.
47. O’Keefe RT, Norman C, Newman AJ. The invariant
U5 snRNA loop 1 sequence is dispensable for the first
catalytic step of pre-mRNA splicing in yeast. Cell 1996;
86:679-89.
48. Schmitt ME, Brown TA, Trumpower BL. A rapid
and simple method for preparation of RNA from
Saccharomyces cerevisiae. Nucl Acids Res 1990; 18:3091-
2.
49. Clark TA, Sugnet CW, Ares M Jr. Genomewide analysis
of mRNA processing in yeast using splicing-specific
microarrays. Science 2002; 296:907-10.
50. Bousquet-Antonelli C, Presutti C, Tollervey D.
Identification of a regulated pathway for nuclear pre-
mRNA turnover. Cell 2000; 102:765-75.
51. Lopez PJ, Séraphin B. YIBD: the Yeast Intron DataBase.
Nucleic Acids Res 2000; 28:85-6.
25. Maquat LE, Carmichael GG. Quality control of
mRNA function. Cell 2001; 104:173-6.
26. Schell T, Kulozik AE, Hentze MW. Integration of splic-
ing, transport and translation to achieve mRNA qual-
ity control by the nonsense-mediated decay pathway.
Genome Biol Reviews 2002; 3:1006.
27. Maquat LE. Nonsense-mediated mRNA decay in
mammals. J Cell Science 2005; 118:1773-6.
28. Ferraiuolo MA, Lee C-S, Lian LW, Hsu JL, Costa-
Mattioli M, Luo M-J, et al. A nuclear translation-like
factor eIF4AIII is recruited to the mRNA during splic-
ing and functions in nonsense-mediated decay. PNAS
2004; 101:4118-23.
29. Fortes P, Inada T, Preiss T, Hentze M, Mattaj IW, Sachs
AB. The yeast nuclear cap binding complex can interact
with translation factor eIF4G and mediate translation
initiation. Mol Cell 2000; 6:191-6.
30. Iborra FJ, Jackson DA, Cook PR. Coupled transcrip-
tion and translation within nuclei of mammalian cells.
Science 2001; 293:1139-42.
31. Dahlberg JE, Lund E, Goodwin EB. Nuclear transla-
tion: What is the evidence? RNA 2003; 9:1-8.
32. Nathanson L, Xia T, Deutscher MP. Nuclear protein
synthesis: A reevaluation. RNA 2003; 9:9-13.
33. Baron-Benhamou J, Fortes P, Inada T, Preiss T, Hentze
MW. The interaction of the cap-binding complex
(CBC) with eIF4G is dispensable for translation in
yeast. RNA 2003; 9:654-62.
34. Fromont-Racine M, Rain J-C, Legrain P. Towards a
functional analysis of the yeast genome through exhaus-
tive two-hybrid screens. Nat Genetics 1997; 16:277-
82.
35. Ho Y, Gruhler A, Heilbut A, Bader GD, Moore L,
Adams SL, et al. Systematic identification of protein
complexes in Saccharomyces cerevisiae by mass spec-
trometry. Nature 2002; 415:180-3.
36. Tarun SZ Jr, Sachs AB. Association of the yeast
poly(A)-tail binding protein with translation initiation
factor eIF-4G. EMBO J 1996; 15:7168-77.
37. Mader S, Lee H, Pause A, Sonenberg N. The transla-
tion initiation factor eIF-4E binds to a common motif
shared by the translation factor eIF-4gamma and the
translational repressors 4E-binding proteins. Mol Cell
Biol 1995; 15:4990-7.
38. Barrass JD, Beggs JD. Splicing goes global. Trends
Genet 2003; 19:295-8.
13. Fortes P, Kufel J, Fornerod M, Polycarpou-Schwarz M,
Lafontaine D, Tollervey D, Mattaj IW. Genetic and
physical interactions involving the yeast nuclear cap-
binding complex. Mol Cell Biol 1999; 19:6543-53.
14. Gingras AC, Raught B, Sonenberg N. eIF4 initiation
factors: effectors of mRNA recruitment to ribosomes
and regulators of translation. Annu Rev Biochem 1999;
68:913-63.
15. Dominguez D, Kislig E, Altmann M, Trachsel H.
Structural and functional similarities between the
central eukaryotic initiation factor (eIF)4A-binding
domain of mammalian eIF4G and the eIF4A-binding
domain of yeast eIF4G. Biochem J 2001; 355:223-30.
16. Hentze M. eIF4G: a multipurpose ribosome adapter.
Science 1998; 275:500-1.
17. Ponting CP. Novel eIF4G domain homologues linking
mRNA translation with nonsense-mediated mRNA
decay. Trends Biochem Sci 2000; 25:423-6.
18. Aravind L, Koonin EV. Eukaryote-specific domains in
translation initiation factors: implications for transla-
tion regulation and evolution of the translation system.
Genome Res 2000; 10:1172-84.
19. Goyer C, Altmann M, Lee HS, Blanc A, Deshmukh M,
Woolford JL Jr, et al. TIF4631 and TIF4632: Two yeast
genes encoding the high-molecular-weight subunits of
the cap-binding protein complex (eukaryotic initiation
factor 4F) contain an RNA recognition motif-like
sequence and carry out an essential function. Mol Cell
Biol 1993; 13:4860-74.
20. McKendrick L, Thompson E, Ferreira J, Morley SJ,
Lewis JD. Interaction of eukaryotic translation initia-
tion factor 4G with the nuclear cap-binding complex
provides a link between nuclear and cytoplasmic func-
tions of the m(7)guanosine cap. Mol Cell Biol 2001;
21:3632-41.
21. Lejeune F, Ranganathan AC, Maquat LE. eIF4G is
required for the pioneer round of translation in mam-
malian cells. Nat Struct Mol Biol 2004; 11:992-1000.
22. Ishigaki Y, Li X, Serin G, Maquat LE. Evidence for a
pioneer round of mRNA translation: mRNAs subject
to nonsense-mediated decay in mammalian cells are
bound by CBP80 and CBP20. Cell 2001; 106:607-
17.
23. Lejeune F, Ishigaki E, Li X, Maquat LE. The exon
junction complex is detected on CBP80-bound but not
eIF4E-bound mRNA in mammalian cells: Dynamics of
mRNP remodeling. EMBO J 2002; 21:3536-45.
24. Hentze MW, Kulozik AE. A perfect message: RNA
surveillance and nonsense-mediated decay. Cell 1999;
96:307-10.
