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Analysis of synthetic lethality reveals genetic interactions
between the GTPase Snu114p
and snRNAs in the catalytic core of the Saccharomyces cerevisiae
spliceosome
Lily Novak Frazer, Simon C. Lovell and Raymond T. O’Keefe*
The University of Manchester, Faculty of Life Sciences, Michael
Smith Building, Manchester, United Kingdom, M13 9PT
Genetics: Published Articles Ahead of Print, published on July
20, 2009 as 10.1534/genetics.109.107243
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Frazer et. al. page 2
Running Title: SNU114 and snRNA genetic interactions
Key words: pre-mRNA splicing, snRNA, Snu114, Saccharomyces
cerevisiae
*Corresponding author: Dr. Raymond T. O’Keefe The University of
Manchester Faculty of Life Sciences Michael Smith Building Oxford
Road, Manchester M13 9PT United Kingdom Tel: 44 161 275 7393 Fax:
44 161 275 5082 Email: [email protected]
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Frazer et. al. page 3
ABSTRACT
Conformational changes of snRNAs in the spliceosome required for
pre-mRNA splicing are
regulated by eight ATPases and one GTPase Snu114p. The Snu114p
guanine state regulates U4/U6
unwinding during spliceosome activation and U2/U6 unwinding
during spliceosome disassembly
through the ATPase Brr2p. We investigated 618 genetic
interactions to identify an extensive
genetic interaction network between SNU114 and snRNAs. Snu114p G
domain alleles were
exacerbated by mutations that stabilize U4/U6 base pairing. G
domain alleles were made worse by
U2 and U6 mutations that stabilize or destabilize U2/U6 base
pairing in helix I. Compensatory
mutations that restored U2/U6 base pairing in helix I relieved
synthetic lethality. Snu114p G
domain alleles were also worsened by mutations in U6 predicted
to increase 5’ splice site base
pairing. Both N-terminal and G domain alleles were exacerbated
by U5 loop 1 mutations at
positions involved in aligning exons while C-terminus alleles
were synthetically lethal with U5
internal loop 1 mutations. This suggests a spatial orientation
for Snu114p with U5. We propose
that the RNA base pairing state is directly or indirectly sensed
by the Snu114p G domain allowing
the Snu114p C-terminal domain to regulate Brr2p or other
proteins to bring about RNA/RNA
rearrangements required for splicing.
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Frazer et. al. page 4
INTRODUCTION
Introns in pre-messenger RNA (pre-mRNA) are removed by the
spliceosome to yield mature
mRNA which is then exported into the cytoplasm for translation.
The process of pre-mRNA
splicing has been reviewed extensively (MOORE et al. 1993; WILL
and LÜHRMANN 2006). Splicing
is carried out in the nucleus by the assembly of small nuclear
ribonucleoprotein particles (or
snRNPs) onto the pre-mRNA and occurs by two transesterification
steps which are carried out
within the spliceosome by a series of RNA:RNA interactions and
RNA:protein rearrangements.
Five snRNPs take part in splicing, U1, U2, U4, U5 and U6, of
which the last three form a tri-snRNP
in which U4 is base paired with U6 (MOORE et al. 1993). Although
it is thought that the RNAs
within snRNPs are responsible for catalyzing the two steps of
splicing, it is the many proteins, in
particular the NTPases, which bring about the multiple
rearrangements that are required for
catalysis by the snRNAs (SMITH et al. 2008). Key remodelling
steps during splicing include
unwinding of U4/U6 helices, formation of U2/U6 interactions,
unwinding of U2/U6 helices and re-
establishment of U4/U6 complexes. Remodelling is facilitated by
eight ATPases and one GTPase,
Snu114p, one of the key protein factors required for the
regulation of spliceosomal dynamics
(reviewed in (FRAZER et al. 2008).
The single GTPase of the spliceosome, Snu114p (U5-116kDa or
hSnu114 in humans), is a
protein of the U5 snRNP (FABRIZIO et al. 1997). It shares
significant sequence homology with
translation elongation factor 2 (EF-2) and contains five domains
similar to EF-2, with domain I
containing the G domain consensus sequence elements G1–G5 that
are important for binding and
hydrolysis of GTP. hSnu114 has been found to crosslink to GTP
specifically and a mutation in
Snu114p, expected to abolish GTP binding, is lethal, suggesting
that GTP binding and hydrolysis
are necessary for Snu114p function in vivo (BARTELS et al. 2003;
FABRIZIO et al. 1997). Snu114p
also has a unique acidic amino-terminus (N-terminus) which is
implicated, along with the G
domain, in U4/U6 unwinding during spliceosome assembly (BARTELS
et al. 2002).
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Frazer et. al. page 5
The relationship of Snu114p to other spliceosomal proteins has
been investigated using
conditionally lethal alleles to test for genetic interactions
with other splicing factors (BRENNER and
GUTHRIE 2005). Genetic interactions of snu114 alleles were found
with prp8, brr2, prp28, prp19,
sad1 and snu66 alleles. The genetic interactions of SNU114 with
PRP8 and BRR2 were expected
considering the proteins form a stable complex within the U5
snRNP in human cells (ACHSEL et al.
1998). Other genetic interactions were with genes that are all
known to act before the first step of
splicing, suggesting an important role for Snu114p during
spliceosomal activation (BRENNER and
GUTHRIE 2005). Although genetic interactions do not necessarily
indicate direct interactions, two
hybrid studies have confirmed interactions between hSnu114,
hPrp8 and hBrr2 (LIU et al. 2006)
and other studies have indicated a requirement for Prp8p and
Snu114p for correct U5 snRNP
assembly (BRENNER and GUTHRIE 2006). Whether Snu114p function
requires GTP hydrolysis was
addressed by analysis of spliceosome assembly and disassembly,
both of which were promoted by
Snu114p and Brr2p (SMALL et al. 2006). The guanine nucleotide
state of Snu114p, rather than GTP
hydrolysis, regulated Brr2p activity during snRNA unwinding.
Compared to the number of known Snu114p-protein interactions,
little is known about how
Snu114p interacts with snRNAs of the spliceosome. Evidence of
direct Snu114p-RNA interactions
include hSnu114 crosslinks to hairpins introduced in pre-mRNA
and hSnu114 crosslinks with the
pre-mRNA pyrimidine tract after spliceosome assembly but prior
to the second step (CHIARA et al.
1997; LIU et al. 1997). This suggested a role for hSnu114 in
bringing the 3’ splice site into the
active site for the second step of splicing. Parallels have been
drawn between the function of
Snu114p in the spliceosome and EF-2 in the ribosome (STALEY and
GUTHRIE 1998). The
interaction between U5 loop 1 and the 5’-exon during splicing
was proposed to be analogous to the
tRNA anticodon–codon interaction during translation, with a
conformational change driving
Snu114p to reposition the 5’-exon-bound U5 loop 1 for the second
step of splicing (STALEY and
GUTHRIE 1998). Although Snu114p was found to crosslink to the 5’
side of internal loop 1 of U5
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Frazer et. al. page 6
snRNA (DIX et al. 1998), a model for Snu114p function based on
GTP driven hydrolysis is no
longer supported (SMALL et al. 2006).
As splicing is most likely RNA-catalyzed, it is important to
determine any interactions
between Snu114p and the snRNAs involved in catalysis. We
assessed synthetic lethal interactions
of SNU114 with the snRNAs that contribute to the catalytic core
of the spliceosome, including U4
which dissociates from the spliceosome prior to assembly of the
active complex. We found that
snu114 G domain mutations were synthetically lethal with
mutations in U4 that stabilize the upper
portion of U4/U6 stem I and only one mutation within stem I.
Similarly, snu114 G domain
mutations were synthetically lethal with mutations in U2 and U6
predicted to stabilize or destabilize
U2/U6 base pairing in helix I. Synthetic lethality could be
suppressed by compensatory mutations
that restored U2/U6 base pairing in helix I. In addition,
synthetically lethal interactions of G
domain mutations of snu114 with U6 snRNA alleles indicated a
role for Snu114p during U6 base
pairing interactions with the 5’ splice site. These results
suggest that the Snu114p G domain is
important for sensing the integrity of RNA/RNA interactions in
the catalytic core of the
spliceosome. The role of Snu114p within the catalytic core was
confirmed by genetic analysis with
U5. We found that snu114 N-terminal and G domain mutations were
synthetically lethal with U5
loop 1 alleles in positions important for tethering the exons
during splicing and that C-terminal
mutations were synthetically lethal with alleles in U5 internal
loop 1. These results suggest a
spatial orientation for Snu114p with U5 and a possible model for
regulation. The Snu114p G
domain, and to some extent the N-terminal domain, may sense the
state of the snRNAs in the
catalytic core and, possibly through a structural rearrangement
with U5, transmit this information to
the C-terminal domain to regulate Brr2p or other proteins to
bring about the rearrangements in the
spliceosome required for splicing.
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Frazer et. al. page 7
MATERIALS AND METHODS
Yeast strains and manipulation
For analysis of SNU114 mutations, diploid yeast strain Y25023
(Table 1) was obtained from
EUROSCARF and transformed with pRS416-Snu114. All
transformations were carried out using
the lithium acetate procedure (GIETZ et al. 1992).
G418-resistant and Ura+ transformants were
sporulated and tetrads dissected. Haploid progeny were checked
for disruption of SNU114 with
kanMX4 by PCR of genomic DNA to identify the strains YSNU114KO1
and YSNU114KO2 (Table
1). SNU114 knockout strain, YSNU114KO1, containing pRS416-Snu114
was transformed with
each snu114 allele on a separate plasmid. Transformants were
transferred to 5-fluoro-orotic acid
(5FOA) to select against the URA3 plasmid by plasmid shuffle
(BOEKE et al. 1984) and to
determine the viability of snu114 alleles as the sole source of
Snu114p. Cells viable on 5FOA were
tested for temperature sensitivity by streaking onto yeast
peptone dextrose (YPD) agar and
incubating at 16°, 25°, 30°, 37°, 39° and 40° for 3-10 days.
To test genetic interactions between snu114 and snRNA alleles,
double deletion strains were
constructed by mating strain YSNU114KO2 with strain YU5KO
(O'KEEFE 2002), YU2KO or
YU6KO (Table 1). Mated diploid strains were transferred to 5FOA
to generate diploid strains
lacking pRS416 plasmids that were complementing the SNU114 and
snRNA knockouts. Diploid
strains with single SNU114 and individual snRNA deletions were
transformed with a pRS416
plasmid containing both WT genes (pRS416-Snu114-U5,
pRS416-Snu114-U2 or pRS416-Snu114-
U6). Viable G418-resistant and Ura+ transformants were
sporulated and tetrads dissected. The
haploid progeny were analyzed for disruption of SNU114 and the
relevant snRNA gene by PCR of
genomic DNA and by plasmid shuffle for the requirement of
Snu114p and U5, U2 or U6 to identify
the strains YSNU114/U5KO, YSNU114/U2KO and YSNU114/U6KO (Table
1). YSNU114/U4KO
was constructed by replacing the U4 gene with the hphNT1
resistance gene (JANKE et al. 2004) in
the SNU114 diploid knockout strain Y25023 with a PCR product
amplified from pYM24 using
primers U4hphNT1F and U4hphNT1R (Table S1). G418- and hygromycin
B-resistant cells were
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Frazer et. al. page 8
transformed with pRS416-Snu114-U4, sporulated and tetrads
dissected. Haploid progeny were
checked for integration of kanMX4 and hphNT1 by PCR of genomic
DNA and in vivo for the
requirement of both Snu114p and U4 to identify the strains
YSNU114/U4KO1 and
YSNU114/U4KO2 (Table 1). Strains YSNU114/U5KO, YSNU114/U2KO,
YSNU114/U6KO and
YSNU114/U4KO1 (Table 1) were transformed with snu114 and snRNA
alleles on different
plasmids and then tested for synthetic lethality by plasmid
shuffle.
Plasmid construction and mutagenesis
SNU114 was cloned by PCR amplification using Pfu DNA polymerase
(Stratagene) of yeast
genomic DNA (Promega) using primers Snu114F and Snu114B (Table
S1) complementary to
regions upstream and downstream of the open reading frame and
containing restriction sites for
EcoRI and BamHI. The PCR product was digested with EcoRI and
BamHI, then ligated into
pRS416 (SIKORSKI and HIETER 1989) to produce pRS416-Snu114.
pRS416 plasmids containing
SNU114 and each snRNA gene were made in the following manner.
pRS314-U5 (m571) (O'KEEFE
et al. 1996) was digested with EcoRI/XhoI and the U5 gene
fragment was ligated into EcoRI/XhoI
digested pRS416-Snu114 to generate pRS416-Snu114-U5. pRS415-U2
(MCGRAIL et al. 2006) was
digested with BamHI, filled in with T4 DNA polymerase (Roche),
then digested with XhoI. The U2
gene fragment was ligated into pRS416-Snu114 digested with
HindIII, filled in as above, then
digested with XhoI to generate pRS416-Snu114-U2. pRS413-U6
(MCGRAIL et al. 2006) was
digested with SacI, blunt ended with T4 DNA polymerase, then
digested with XhoI. The U6 gene
fragment was ligated into pRS416-Snu114p digested with HindIII,
filled in as above, then digested
with XhoI to generate pRS416-Snu114-U6. pRS416-Snu114-U4 was
constructed by PCR
amplification of the U4 gene using primers U4GF and U4GR
containing restriction sites for BamHI
and KpnI (Table S1). The PCR product was digested with BamHI and
KpnI, then ligated into
pRS416-Snu114 to produce pRS416-Snu114-U4. The PCR product was
also digested with BamHI
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Frazer et. al. page 9
and ligated into a BamHI/HincII digested pRS413 to produce
pRS413-U4. The identity of all
plasmids was confirmed by sequencing.
Conditionally lethal snu114 alleles were generated by
identifying hydrophobic stretches of
amino acids in an alignment of SNU114 with the yeast elongation
factor, EFT2. Amino acids
within hydrophobic regions were mutated individually to aspartic
acid. Lethal substitutions
indicated residues that were good candidates for conditional
lethality (CHAKSHUSMATHI et al.
2004). To generate conditionally lethal mutations amino acids
within the hydrophobic regions of
Snu114p were mutated individually to alanine, serine, lysine or
tryptophan. pBK413-Snu114 used
for mutagenesis was constructed by digesting pRS416-Snu114 with
EcoRI and BamHI, then
ligating the SNU114 gene fragment into pBK413 (FRAZER and
O'KEEFE 2007). Mutagenesis of
pBK413-Snu114 was by the method of Kunkel (KUNKEL 1985), using
the primers listed in Table
S1. Plasmids containing snu114 mutations were named according to
the amino acid substitution
introduced (e.g. V266P, P860K, see Table 2). The snu114
N-terminal deletion mutation (ΔN),
which lacked the first 128 amino acids of SNU114, was made as
described previously (BARTELS et
al. 2002). Mutations snu114-12, -14, -15, -30 and -40 (P216N,
L381P, K146I, G646R and M842R
respectively) were described by Brenner and Guthrie (2005).
Selected temperature sensitive snu114
mutations in pBK413-Snu114 were transferred to pRS415.
Plasmids pRS314-U5, pRS415-U2, pRS413-U6 and pRS413-U4 were
mutagenized as
described above, using the primers listed in Table S1. Plasmids
containing mutated U5, U2, U6 or
U4 were named according to the mutation introduced (see Table
3). The identity of all mutations
was confirmed by sequencing.
The three-dimensional model of Snu114p was constructed by
comparative modelling. Yeast
elongation factor 2 (Eft2p, pdb code 1n0v) (JORGENSEN et al.
2003) was selected as the template
structure as it was the best sequence match based on BLAST score
(ALTSCHUL et al. 1990). The
sequences of Snu114p and Eft2p were aligned using ClustalW
(LARKIN et al. 2007). The N-
terminal domain (residues 1-121) and a region from the
C-terminus (residues 982-1008) were
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Frazer et. al. page 10
omitted from the model as there are no equivalent residues in
the crystal structure of Eft2p. The
percentage sequence identity over the aligned region was 25.3%.
Regions of buried hydrophobic
amino acids within the structure of Eft2p were identified using
Modeller (SALI and BLUNDELL
1993) and corresponding homologous regions in Snu114p were then
identified and targeted for
mutagenesis. Ten three dimensional models were built using
Modeller (SALI and BLUNDELL 1993)
and the model with lowest pseudo-energy (that when combined best
fit to all input data used to
generate the model) was chosen.
Snu114 protein levels
To analyze protein levels, overnight YPD cultures of cells
containing WT or mutated snu114 as the
only source of Snu114p were used to inoculate YPD cultures to an
OD600 of 0.5 and grown for at
least three generations at permissive (30°) or restrictive (39°)
temperatures. Cells were sedimented
by centrifugation and washed with water prior to being lysed in
SDS-loading buffer with acid-
washed glass beads (Sigma) by vigorous vortexing. Cells were
boiled for five minutes prior to
centrifugation to remove cell debris and extracts removed then
separated on 12% acrylamide
PAGEr gels (Lonza). Levels of Snu114p were detected by western
blots with rabbit anti-Snu114p
antibodies generated against a peptide encompassing the first 15
amino acids of Snu114p and
standardized by probing with mouse anti-Zwf1p antibodies
(Sigma).
Primer extensions
To determine whether snu114 strains exhibited splicing defects,
total RNA was extracted with hot
phenol (KOHRER and DOMDEY 1991) from strain YSNU114KO1 in the
presence of pBK413-based
WT or mutated snu114 after incubation in YPD for 16 h at the
restrictive temperature of 39°. Equal
amounts of total RNA was hybridized to a 32P end-labelled
oligonucleotide primer, U3snoRNA-RT
(Table S1), and primer extensions were carried out as described
previously (DOBBYN and O'KEEFE
2004).
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Frazer et. al. page 11
Genetic analysis of synthetic lethality
YSNU114/U5KO was transformed with mutated pBK413-Snu114 and
pRS414-U5 and
transformants selected on synthetic defined (SD) medium
(SD-Ura-His-Trp) (BIO 101 Systems).
YSNU114/U2KO was transformed with mutated pRS413-Snu114 and
pRS415-U2,
YSNU114/U6KO with mutated pRS415-Snu114 and pRS413-U6 and
YSNU114/U4KO1 with
mutated pRS415-Snu114 and pRS413-U4 and the transformants
selected on SD-Ura-His-Leu.
Transformants were then tested for synthetic lethality by
plasmid shuffle on 5FOA. Synthetic
lethality was scored as the lack of growth after 3 days at 30°.
Synthetic sickness was scored as
growth of smaller or fewer colonies compared to control sectors
after 3 days at 30°.
Compensatory analysis
To determine whether the synthetic lethality observed between
snu114 and snRNA mutations could
be alleviated, synthetically lethal combinations were
transformed with a plasmid containing an
snRNA mutation that restored base pairing in U4/U6 stem I or
U2/U6 helices. This was achieved
by producing a vector with MET15 as the selection marker. A 2491
base pair fragment containing
MET15 (COST and BOEKE 1996) was amplified by PCR from yeast
genomic DNA with primers
Met15F and Met15B (Table S1). The Met15F primer contained AfeI
and the Met15B primer
contained BamHI and PstI restriction enzyme recognition sites.
The PCR product was digested
with BamHI and cloned into pRS414 digested with HincII and
BamHI. pRS414 plasmids
containing the MET15 fragment were tested for complementation of
MET15 in YSNU114/U4KO2.
The MET15 was released from a complementing plasmid with AfeI
and PstI, then cloned into
pRS413 where most of the HIS3 gene had been removed by digestion
with AfeI and NsiI to produce
the plasmid pRS413MET15. Mutations in the U2, U4 and U6 snRNAs
were transferred into this
plasmid.
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Frazer et. al. page 12
To assess suppression of Snu114-U2 synthetic lethality,
YSNU114/U2KO was transformed
with synthetically lethal mutation combinations of pBK413-Snu114
and pRS415-U2, as well as
pRS413MET15 containing U6 mutations to restore U2/U6 base
pairing in the presence of wild type
U6. To assess suppression of Snu114-U4 synthetic lethality,
YSNU114/U4KO2 was transformed
with synthetically lethal combinations of pRS415-Snu114 and
pRS413-U4, as well as
pRS413MET15 containing a U6 mutation to restore base pairing in
U4/U6 in the presence of wild
type U6. To assess suppression of Snu114-U6 synthetic lethality,
YSNU114/U6KO was
transformed with synthetically lethal mutation combinations of
pRS415-Snu114 and pRS413-U6, as
well as pRS413MET15 containing U2 mutations to restore U2/U6
base pairing or U4 mutations to
restore U4/U6 base pairing in the presence of wild type U2 or
U4. Transformants were selected on
SD-Ura-His-Leu-Met agar. Transformants were then tested for
growth by plasmid shuffle on 5FOA
alongside the synthetically lethal combinations and controls.
Suppression of synthetic lethality was
scored after 3 days at 30°.
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Frazer et. al. page 13
RESULTS
Identification of additional snu114 mutations that are
temperature sensitive and show splicing
defects at the first step of splicing
Forty-nine single and four double amino acid substitution
mutations were generated within
five predicted hydrophobic regions of Snu114p: L234-I239,
C264-I267, I311-A314 in domain I,
I561-I564 in domain II, and P860-I861 in domain IVa. Nineteen
mutations were lethal and of the
viable alleles five were temperature sensitive (Ts) at 39°
(V238D, V266P, F313Q, L563P and
P860K) and one was Ts at 37° (L563F) (Table 2). The Ts mutations
mapped to different locations
within Snu114p (Figure 1A). The V238D mutation was proximal to
the putative salt bridge at
D233 (with R488) and the V266P mutation was near the critical
(N/T)(K/Q)XD G4 motif. This
motif is required for GTP binding and hydrolysis (FABRIZIO et
al. 1997) and stabilizing residues in
the G1 motif (BOURNE et al. 1991). The F313Q mutation was
adjacent to the (G/T)SAL G5 motif,
which stabilizes residues in the G4 motif (BOURNE et al. 1991),
L563F was within domain II and
P860K was in domain IVa (Figure 1A). Previously published Ts
snu114 alleles (K146I, P216N,
L381P, G646R and M842R) (BRENNER and GUTHRIE 2005) are
positioned in different locations
within the predicted structure from the Ts alleles generated in
this study (Figure 1B). An N-
terminal snu114 deletion mutation lacking the first 128 amino
acids (ΔN) (BARTELS et al. 2002)
was also produced (Table 2). For clarity, snu114 alleles are
annotated with their putative positions
within Snu114p, for example, V266P (G4) signifies the mutation
is in G domain motif G4 and
P860K (IVa) signifies the mutation is in domain IVa.
To determine whether the temperature sensitivity of snu114
alleles was due to defects in
protein expression or in pre-mRNA splicing, a SNU114 knockout
strain carrying WT or snu114
allele on a plasmid was grown at the permissive and restrictive
temperatures. Four strains chosen
for analysis were V266P (G4), F313Q (G5), L563F (II) and P860K
(IVa) (Figure 2A) as these were
the most reliable Ts mutations that spanned the predicted three
dimensional structure of Snu114p
(Figure 1B). Protein levels in non-Ts (WT and V266K (G4)) and Ts
strains were equivalent at the
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Frazer et. al. page 14
permissive temperature as demonstrated by western blotting
(Figure 2B, lanes 1-7). When cells
were grown at the restrictive temperature for at least three
generations, protein levels remained
constant in WT (Figure 2B, lane 8), non-Ts (Figure 2B, lane 9)
and Ts strains (Figure 2B, lanes 10-
11, 13-14). The exception was the mutation L563P (II) (Figure
2B, lanes 5 and 12), which
displayed a slight decrease in Snu114p levels compared to WT at
both permissive and restrictive
temperatures so was excluded from further investigations. Thus
the Ts phenotype observed in
snu114 mutant strains was not due to aberrant levels of protein
expression. To assess whether the
temperature sensitivity of snu114 mutant strains was due to a
defect in pre-mRNA splicing, total
RNA was extracted from strains grown overnight at the
restrictive temperature. The levels of
spliced and unspliced U3A and U3B snoRNAs were analyzed by
primer extension with a primer
complementary to their common exon 2. In WT extracts, only a
small amount of unspliced U3A or
U3B precursor was detectable (Figure 2C, lane 3). In extracts
from snu114 mutant strains, there
was a significant accumulation of unspliced U3A and U3B
precursor (Figure 2C, lanes 4-7)
compared to levels in WT extracts, indicating that splicing was
inhibited and that the block in
splicing was prior to the first step. Note that a second step
block could not be detected by primer
extension in this manner.
snu114 mutations display synthetic lethality with U4 snRNA
mutations that alter U4/U6 base
pairing
The interactions of U4 with U6 serve to regulate the U6 snRNA
during assembly and
activation of the spliceosome. Unwinding of U4 from U6 during
spliceosome assembly is catalyzed
by the DExD/H-box helicase Brr2p (LAGGERBAUER et al. 1998;
RAGHUNATHAN and GUTHRIE
1998) which is regulated by the guanine nucleotide state of
Snu114p (SMALL et al. 2006). It is not
clear whether the regulation of Brr2p activity by Snu114p is
through direct or indirect interactions
with Brr2p, another protein, or the U4 or U6 snRNAs. Genetic
interactions were investigated
between snu114 alleles and mutations in U4 positions that base
pair with U6 snRNA in stem I and
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Frazer et. al. page 15
II of the U4/U6 complex and U4 mutations adjacent to stem I (HU
et al. 1995; LI and BROW 1996;
SHANNON and GUTHRIE 1991) as shown in Figure 3. A mutation
adjacent to U4 stem I, U4-cs1
(A66U,A67U,A68G), stabilizes U4/U6 stem I by increasing base
pairing with the U6 ACAGAGA
region thereby blocking U4/U6 dissociation early in splicing
(KUHN et al. 1999; LI and BROW
1996). Genetic analysis revealed that K146I (G1), V266P (G4) and
F313Q (G5) demonstrated
synthetic lethality with U4-cs1 (Figure 3 and Table 4).
Interestingly, P216N (G2) and L381P (G’’)
were not synthetically lethal but noticeably sick with U4-cs1
(Figure 3 and Table 4). In order to
analyze the effects of other mutations that extend base pairing
in the same region of stem I as U4-
cs1, mutations U4-U64C, U4-G65A and U4-U64C,G65A were tested for
synthetic lethality with
snu114 mutations. Genetic analysis revealed that K146I (G1) was
synthetically lethal with the
double mutation U4-U64C,G65A and synthetically sick with U4-U64C
and U4-G65A (Figure 3 and
Table 4). Therefore, it appears that extending U4/U6 base
pairing in stem I results in synthetic
lethality with snu114 G domain mutations.
Although many positions in U4 stem I have been shown to be
tolerant to mutation, only U4-
G58 has been shown to have any phenotypic effects (HU et al.
1995). Analysis of genetic
interactions between mutations in snu114 and U4 stem I (U57A,
G58A, C59U, U60C, U64G)
revealed that only K146I (G1) was synthetically lethal with
U4-G58A (Figure 3 and Table 4). No
other snu114 alleles were synthetically lethal with U4-G58A
(Table 4) although V266P (G4),
F313Q (G5), G646R (III) and M842R (IVa) were synthetically sick
(Figure 3 and Table 4). While
the U4-U57A, U4-G58A, U4-C59U, U4-U60C and U4-U64G mutations are
all predicted to disrupt
base pairing between U4 and U6, U4-G58A is the only mutation
that disrupts stem I leading to a Ts
phenotype (HU et al. 1995) and displays synthetic lethality and
sickness with snu114 mutations in
the G domain and domains III and IVa. These results indicate
that only mutation at this nucleotide
of U4 in stem I exacerbates snu114 mutations.
The U4 stem II position G14 is responsible for U4 snRNA
functions early in splicing (HU et
al. 1995). Analysis of genetic interactions between snu114
alleles and U4-G14C, which disrupts
-
Frazer et. al. page 16
U4/U6 stem II base pairing, thus destabilizing the U4/U6 complex
(SHANNON and GUTHRIE 1991;
WERSIG and BINDEREIF 1990), revealed that no snu114 mutation
displayed synthetic lethality with
this U4 mutation (Table 4).
snu114 mutations display synthetic lethality with U6 snRNA
mutations important for base
pairing with the 5’ splice site and that alter U2/U6 base
pairing
The role of U6 snRNA is pivotal in the spliceosome as it is
involved in three important base
pairing interactions during splicing: with U4 in the U4/U6
di-snRNP (MADHANI et al. 1990), with
the 5’ splice site via a conserved single-stranded ACAGAGA
(A47-A53) sequence following tri-
snRNP incorporation into the spliceosome (MADHANI et al. 1990;
SAWA and SHIMURA 1992;
SONTHEIMER and STEITZ 1993) and with U2 snRNA in helix I in the
active site before the first step
of splicing (MADHANI and GUTHRIE 1992). As Snu114p regulates
Brr2p activity and, as shown
above, interacts genetically with mutations in U4 that perturb
the U4/U6 complex we determined
whether SNU114 interacts genetically with U6.
Genetic interactions were investigated between snu114 alleles
and mutations in the U6
ACAGAGA sequence at positions A47, G50 and G52. The analysis
revealed synthetic lethality
between V266P (G4) and U6-G50U and U6-G52U as well as synthetic
lethality between F313Q
(G5) and U6-A47G, U6-G50U and U6-G52U (Figure 4 and Table 5).
Although L563F (II) and
M842R (IVa) were synthetically sick with U6-A47G, and K146I was
synthetically sick with U6-
G50U and U6-G52U, no other snu114 mutations were synthetically
lethal/sick with U6
ACAGAGA mutations (Table 5). Interestingly, U6-G50U and U6-G52U
both potentially increase
the base pairing of U6 with the conserved 5’ splice site
sequence. Therefore, it appears that
stabilization of the interaction of U6 with the 5’ splice site
exacerbates snu114 G domain mutations.
Although U6-A53 is included in the ACAGAGA sequence, the
mutation U6-A53U is
expected to extend the base pairing between U2 and U6 in helix
I. Genetic interactions with this U6
mutation showed that K146I (G1), V266P (G4) and F313Q (G5) were
synthetically lethal and
-
Frazer et. al. page 17
L381P (G’’) was synthetically sick (Figure 4). This suggests
that extending U2/U6 helix Ia
exacerbates snu114 G domain mutations.
Genetic interactions were also investigated between snu114
mutations and U6 insertion
mutations at positions A53/U54 and G55/A56 to determine whether
extending the distance between
the ACAGAGA sequence and U4/U6 stem I or U2/U6 helix I had any
effects. No synthetic
lethality was observed between snu114 alleles and U6 mutations
Ins 1U A53/U54 and Ins 1U
G55/A56 (Table 5). Substitution mutations at U6-G55 and U6-A56
are expected to weaken stem I
by disrupting base pairing between U4 and U6 snRNAs (LI and BROW
1996) and insertion
mutations at U6-G55/A56 have been shown to progressively inhibit
the first step of splicing
(MCGRAIL et al. 2006). Synthetic lethality was observed between
snu114 mutations and
substitution mutations at U6-U54 and U6-G55 which would be
expected to extend the base pairing
at the top of U4/U6 stem I or disrupt the base pairing in U2/U6
helix I. In particular, K146I (G1)
was synthetically lethal with U6-U54C, K146I (G1) and V266P (G4)
with U6-G55A and K146I
(G1) and V266P (G4) with U6-U54C,G55A (Figure 4). In addition,
F313Q (G5) was synthetically
sick with all three substitution mutations U6-U54C, U6-G55A and
U6-U54C,G55A (Table 5). This
suggests that extending the base pairing of U4/U6 at the top of
stem I or disrupting the base pairing
of U2/U6 helix Ia exacerbates snu114 G domain mutations. These
results substantiate the results
obtained with U4-U64C and U4-G65A mutations, which also extend
the base pairing of U4/U6
stem I and result in synthetic lethality with snu114 G domain
mutations (see previous section) and
the results obtained with U2/U6 helix Ia mutations (see
following section).
Further analysis revealed synthetically lethal interactions
between snu114 alleles and U6
mutations in the region that mutually exclusively base pairs
with U4 in stem I prior to spliceosome
activation or with U2 in helix I. Double mutations at U6-A56,U57
are conditionally lethal but can
be suppressed by U2 mutations which restore helix Ia base
pairing (MADHANI and GUTHRIE 1992).
Our analyses revealed that K146I (G1) and V266P (G4) were
synthetically lethal with U6-
A56C,U57C (Figure 4 and Table 5). The ΔN, P216N (G2), F313Q
(G5), L381P (G’’) and M842R
-
Frazer et. al. page 18
(IVa) mutations were also synthetically sick with U6-A56C,U57C
(Figure 4), but no other snu114
alleles were synthetically lethal with U6-A56C,U57C (Table 5).
In addition, the only other genetic
interaction in this U6 region was a synthetically sick
interaction between V266P (G4) and U6-C58U
(Table 5).
Mutations in snu114 and the U6-AGC triad that base pairs with U4
to form stem I in the
U4/U6 complex or with U2 to form helix Ib in the U2/U6 complex
were investigated for synthetic
lethality. In yeast, substitutions in U6-G60 and U6-C61 result
in defects in 5’ splice site cleavage
and substitutions in U6-A59 cause defects in exon ligation and
inhibit the second step of splicing
(FABRIZIO and ABELSON 1990; MCGRAIL et al. 2006). Synthetic
lethality was observed between
both V266P (G4) and F313Q (G5) and U6-A59C (Figure 4 and Table
5). In addition, ΔN and
K146I (G1) were synthetically sick with U6-A59C (Figure 4 and
Table 5). Further analysis
revealed that all snu114 G domain mutations, as well as ΔN and
M842R (IVa), were synthetically
lethal with U6-C61G (Figure 4 and Table 5). No other snu114
alleles were synthetically lethal with
U6-C61G (Table 5). Overall, it appears that snu114 mutations in
the N-terminus and the G domain
are exacerbated by mutations in U6 that disrupt U2/U6 base
pairing within helices Ia and Ib.
Finally, genetic interactions between snu114 and U6-A62C were
investigated to assess
whether disrupting the base pairing with U4-U57 at the base of
U4/U6 stem I or with U6-C85 in U6
internal stem loop (ISL) or whether extending the base pairing
in helix Ib with U2-G20 caused
synthetic lethality. There was no observed synthetic lethality
between U6-A62C and any snu114
mutation (Table 5). This supports the lack of synthetic
lethality of snu114 G domain mutations
with U4-U57A and suggests that Snu114p does not interact
genetically with U6-ISL at position U6-
A62. These data also suggest that although extending the base
pairing of U2/U6 helix Ia with U6
mutations exacerbated snu114 G domain mutations, extending the
base pairing of U2/U6 helix Ib
does not have the same effect.
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Frazer et. al. page 19
snu114 mutations display synthetic lethality with U2 snRNA
mutations that alter U2/U6 base
pairing
Two important base pairing interactions of the U2 snRNA are with
the branchpoint
sequence (BPS) early in spliceosome assembly (MOORE et al. 1993)
and with U6 to form helix Ia
and Ib in the spliceosome active site (MADHANI and GUTHRIE
1992). The U2/U6 helices serve to
bring the 5’ exon together with the BPS adenosine ready for the
first step of splicing (MADHANI and
GUTHRIE 1992). We have shown that mutations in the N-terminal
and G domains of Snu114p
display synthetic lethality with mutations in U6 that increase
base pairing with the 5’ splice site and
disrupt or extend U2/U6 base pairing. Next we determined whether
there were genetic interactions
between mutations in snu114 and the U2 snRNA. Mutations in
snu114 were tested for synthetic
lethality with U2 helix Ib mutations at positions U2-C22 and
U2-U23 (MADHANI and GUTHRIE
1992). U2-C22 forms intramolecular base pairing interactions
with U6-AGC triad nucleotide U6-
G60 of helix Ib. U2-U23 is implicated in 5’ splice site
recognition, base pairs with U6-A59 of helix
Ib before the second step of splicing, crosslinks to the 3’ exon
in the 3’ exon-lariat intermediate and
is important for the second step of splicing (LUUKKONEN and
SÉRAPHIN 1998; MADHANI and
GUTHRIE 1992; MADHANI and GUTHRIE 1994; NEWMAN et al. 1995). Our
analyses revealed
synthetic lethality between V266P (G4) and both mutations at
U2-C22 and U2-U23, while F313Q
(G5) was synthetically sick with both mutations at U2-U23
(Figure 5 and Table 6). No other
snu114 alleles were synthetically lethal with these U2 mutations
(Table 6). Interestingly, there was
no synthetic lethality between any snu114 alleles and several
mutations in the bulge, positions U2-
U24 and U2-A25 which link U2/U6 helix Ia and Ib (Table 6). These
results suggest, similar to that
found with U6 helix Ib mutations, that snu114 G domain mutations
display synthetic lethality with
U2 mutations that disrupt U2/U6 base pairing.
Genetic interactions were also investigated between snu114
alleles and U2-A27C,U28C
positions involved in base pairing with U6 in helix I. A second
U2 mutation in this region, U2-Ins
1U U28/C29, is located in a position where increasing insertions
block the second step of splicing
-
Frazer et. al. page 20
(MCGRAIL et al. 2006; MCPHEETERS and ABELSON 1992). Genetic
analysis revealed that both
V266P (G4) and F313Q (G5) were synthetically lethal with
U2-A27C,U28C (Figure 5 and Table
6). In addition, V266P (G4) was also synthetically lethal with
U2-Ins 1U U28/C29 (Figure 5 and
Table 6). No other snu114 alleles were synthetically lethal with
these U2 mutations (Table 6).
These results suggest that snu114 G domain mutations are
exacerbated by mutations in U2 that
disrupt U2/U6 helix Ia. Further analysis revealed that there was
no synthetic lethality between any
snu114 alleles and insertion mutations U2-Ins 1U A30/A31 and
U2-Ins 1U G32/U33, which are
situated between helix I and the BPS (Table 6). However,
synthetic lethality was observed between
V266P (G4) and U2-A31U and L381P (G’’) and U2-A31U, which
extends the base pairing of
U2/U6 helix Ia (Figure 5). These results support the idea that
mutations in U2 that disrupt or extend
U2/U6 base pairing display synthetic lethality with snu114 G
domain mutations.
snu114 mutations display synthetic lethality with mutations in
U5 snRNA loop 1 and internal
loop 1
U5 snRNA contributes to the catalytic core of the spliceosome
through U5 loop 1 which is
required for tethering the 5’ and 3’ exons for ligation during
the second step of splicing (NEWMAN
and NORMAN 1992; O'KEEFE and NEWMAN 1998). Protein interactions
between Snu114p, Prp8p
and Brr2p are well known (BRENNER and GUTHRIE 2005; LIU et al.
2006) but the only known direct
interaction of Snu114p with an snRNA is a crosslink with U5-C79
of U5 internal loop 1 (IL1),
found at the base of the stem that carries the important U5 loop
1 (DIX et al. 1998). To determine
whether Snu114p displays synthetic lethality with any mutation
in U5, genetic analyses were
carried out with snu114 alleles and twenty-five mutations that
spanned the entire structure of the U5
snRNA.
Ten mutations in U5 loop 1 were tested for genetic interactions
with nine snu114 alleles
(Figure 6A and Table 7). Our analyses revealed that V266P (G4)
was synthetically lethal with
deletion, substitution and insertion mutations in U5 loop 1,
while ΔN was synthetically lethal only
-
Frazer et. al. page 21
with U5 loop 1 double deletion mutations (Figure 6B and Table
7). In particular both V266P (G4)
and ΔN were synthetically lethal with U5-ΔC94,C95 and
U5-ΔU96,U97, nucleotides important for
tethering and aligning the 5’ and 3’ exons during splicing
(NEWMAN and NORMAN 1992; NEWMAN
et al. 1995; O'KEEFE and NEWMAN 1998; SONTHEIMER and STEITZ
1993). V266P (G4) was also
synthetically lethal with U5-A100U,C101G, and U5-Ins 1U G93/C94,
positions opposite each other
in U5 loop 1 (Figure 6A and Table 7). No other snu114 alleles
displayed synthetic lethality with
any of the U5 loop 1 mutations tested (Table 7). Thus, snu114
N-terminal and G domain mutations
are exacerbated by mutations in U5 loop 1.
As Snu114p was shown to crosslink to U5 IL1 (DIX et al. 1998),
genetic interactions
between mutations in snu114 and U5 IL1 were investigated.
Included were eight substitution and
deletion mutations that spanned positions U5-C79 and U5-G80.
Increasingly larger deletions on the
5’ side of U5 IL1 at or near position U5-C79 were progressively
synthetically sick with M842R
(IVa) (Figure 6C and Table 7).
As mutations on the 5’ side of U5 IL1 did not display synthetic
lethality with mutations in
snu114, mutations on the 3’ side of U5 IL1 were investigated.
Three deletion mutations on the 3’
side of IL1, U5-ΔC111, U5-ΔC112,G113 and U5-ΔC111-G113 were
produced. The U5-ΔC111-
G113 mutation was lethal and not studied further (data not
shown). The analysis revealed that only
M842R (IVa) was synthetically lethal with U5-ΔC111 but G646R
(III), M842R (IVa), P860K
(IVa), as well as ΔN, were synthetically lethal with the U5 IL1
allele, U5-ΔC112,G113 (Figure 6D
and Table 7). Significantly, no snu114 G domain mutations
displayed synthetic lethality with any
mutations in U5 IL1 (Table 7). Thus, our results indicate that
snu114 mutations in the N-terminal
domain and domains III and IV exacerbate mutations in the 5’ and
3’ side of U5 IL1 whereas
snu114 mutations in the N-terminal domain and G domain
exacerbate mutations in U5 loop 1.
-
Frazer et. al. page 22
Compensatory mutations that restore base pairing in U2/U6 helix
I suppress synthetic
lethality with snu114 mutations
One prediction of the hypothesis that Snu114p senses the state
of important RNA/RNA
interactions in the spliceosome is that where synthetic
lethality was caused by disruption of base
pairing, restoration of base pairing with a compensatory
mutation would suppress the synthetic
lethality. This would support the theory that the synthetic
lethality was caused by disruption of base
pairing and not an additional effect of the mutations. To test
whether compensatory mutations
could suppress synthetic lethality observed between snu114 and
snRNA mutations predicted to
disrupt base pairing, a plasmid containing a compensatory snRNA
mutation designed to restore base
pairing was transformed with the synthetic lethal combinations.
The compensatory snRNA
mutation was introduced in the presence of the wild-type copy of
that gene.
A number of synthetic lethal combinations found between snu114
and snRNA mutations
predicted to disrupt base pairing were not suppressed by
compensatory mutations designed to
restore base pairing. Synthetic lethality between K146I (G1) and
U4-G58A was not suppressed by
restoring U4/U6 stem I with U6-C61U (data not shown). Synthetic
lethality between snu114
mutations and U6-A56C,U57C was not suppressed by restoring U4/U6
stem I with U4-G62,U63G
(Figure 7A). Synthetic lethality of V266P (G4) and F313Q (G5)
with U6-A59C was not suppressed
by restoring U4/U6 stem I with U4-U60G (Figure 7B). Synthetic
lethality between snu114
mutations and U6-C61G was not suppressed by restoring U4/U6 stem
I with U4-G58C or restoring
U2/U6 helix Ib with U2-G21C (data not shown). Synthetic
lethality of V266P (G4) with U2-C22A
and U2-C22G was not suppressed by restoring U2/U6 helix Ib with
U6-G60U or U6-G60C,
respectively (data not shown). Synthetic lethality of V266P (G4)
with U2-U23C and U2-U23G was
not suppressed by restoring U2/U6 helix Ib with U6-A59G or
U6-A59C, respectively (data not
shown). Synthetic lethality of snu114 mutations with
U2-A27C,U28C was not suppressed by
restoring base pairing in U2/U6 helix Ia with U6-A56G,U57G (data
not shown). Taken together,
these results indicate that synthetic lethality between some
snu114 and snRNA mutations is not
-
Frazer et. al. page 23
solely due to the disruption of base pairing and may suggest an
additional function for these snRNA
positions besides base pairing.
In contrast to the examples given above a number of synthetic
lethal combinations were
suppressed by compensatory mutations. Synthetic lethality
between snu114 mutations and U6-
A56C,U57C was suppressed when base pairing in U2/U6 helix Ia was
restored with U2-
A27G,U28G (Figure 7A). Synthetic lethality of V266P (G4) and
F313Q (G5) with U6-A59C was
suppressed by restoring base pairing in U2/U6 helix Ib with
U2-U23G (Figure 7B). These results
demonstrate that the Snu114p G domain can sense the state of
base pairing in U2/U6 helix Ia and
Ib.
-
Frazer et. al. page 24
DISCUSSION
Our aim was to determine the genetic interactions of SNU114 with
the snRNAs of the spliceosome.
Snu114p is an integral spliceosomal protein that is present
throughout the splicing cycle and is
known to regulate important snRNA interactions required for
activation and disassembly of the
spliceosome. By producing new conditional snu114 alleles and
combining them with known
conditional snu114 alleles, we have identified an extensive
genetic interaction network between
SNU114 and numerous conditional mutations in the U2, U4, U5 and
U6 snRNAs. We propose that
the G-domain of Snu114p senses the state of important RNA/RNA
interactions formed by the
spliceosome (Figure 8).
Synthetic lethality of SNU114 with mutations that extend U4/U6
stem I confirms its role in
U4/U6 unwinding
Brr2p dependent unwinding of U4/U6 base pairing during
spliceosome activation is
regulated by the guanine nucleotide state of Snu114p (SMALL et
al. 2006), therefore, we first
investigated genetic interactions between snu114 and various U4
snRNA mutations. Synthetically
lethal interactions of snu114 mutations in the G domain were
found with the U4 mutations, U4-cs1
and U4-U64C,G65A, which both extend the U4/U6 stem I. In
addition, synthetic lethality was
observed with reciprocal mutations in U6-U54 and U6-G55 which
also extend the base pairing with
U4 in stem I of the U4/U6 complex. These patterns of synthetic
lethality suggest that G domain
residues in Snu114p are involved in U4/U6 unwinding. This
supports work which implicates the
guanine nucleotide state of Snu114p in Brr2p regulation (SMALL
et al. 2006) and work with a
snu114 GTP binding mutation within G domain motif G4 that blocks
U4/U6 unwinding (BARTELS
et al. 2003). That the snu114 N-terminal deletion mutation did
not interact genetically with U4-cs1
or U4-U64C,G65A was surprising since this snu114 allele has been
shown to block U4/U6
unwinding at elevated temperature (BARTELS et al. 2002).
Intriguingly, in another study no genetic
interactions were identified between snu114 G domain alleles and
the cold sensitive BRR2 allele
-
Frazer et. al. page 25
brr2-1, which decreases U4/U6 unwinding activity of Brr2p and is
synthetically lethal with U4-cs1
(BRENNER and GUTHRIE 2005; KUHN and BROW 2000; RAGHUNATHAN and
GUTHRIE 1998).
Rather, the brr2-1 allele is synthetically lethal with a
C-terminal deletion mutation of snu114
(BRENNER and GUTHRIE 2005), an interaction which has been
substantiated by two hybrid assays
(LIU et al. 2006). Therefore, as known genetic and physical
interactions of Snu114p with Brr2p
appear to be outside the G domain of Snu114p, our genetic
interactions of Snu114p with U4 suggest
that the G domain may sense the state of the U4/U6 helix and
signal to Brr2p when to unwind it.
This signal may be transmitted through the domains of Snu114p
and Brr2p that have been shown to
interact (BRENNER and GUTHRIE 2005; LIU et al. 2006) or through
another protein mediator, such as
Prp8p. Many mutations throughout Prp8p suppress the U4-cs1
mutation (KUHN and BROW 2000;
KUHN et al. 1999) and prp8-1 and prp8-brr mutations impair
formation of U5 snRNP and tri-
snRNPs (BROWN and BEGGS 1992; COLLINS and GUTHRIE 1999). Snu114p
has been shown to
interact genetically with these prp8 mutations through its
C-terminal domain (BRENNER and
GUTHRIE 2005). This suggests that Snu114p may directly or
indirectly sense U4/U6 base pairing
with its G domain and signal the state of the U4/U6 helix
through its C-terminus to Brr2p. This
signalling to Brr2p may require a structural rearrangement
between the G domain and C-terminus of
Snu114p and occur either directly or through Prp8p. Recent in
vitro evidence suggests that the C-
terminus of Prp8p is required for the ATP-dependent unwinding of
U4/U6 by Brr2p (MAEDER et al.
2009) and electron microscopy localisation of Snu114p to a hinge
region of the yeast tri-snRNP
suggests Snu114p may promote rearrangements in the tri-snRNP
(HACKER et al. 2008).
snu114 mutations are exacerbated by only one U4/U6 stem I
mutation
Whereas mutations that extend the U4/U6 complex are
synthetically lethal with snu114 G
domain mutations, the U4-G58A mutation, which is Ts and shows a
block in splicing before or at
the first step of splicing (Hu et al., 1995), is synthetically
lethal or sick with snu114 mutations in the
G domain, domain III and domain IVa. The compensatory mutation
U6-C61U that could restore
-
Frazer et. al. page 26
base pairing in U4/U6 stem I with U4-G58A did not rescue
synthetic lethality. This suggests that in
addition to base pairing with U6-C61 in stem I, U4-G58 has
another role during splicing. This role
may be in steps prior to tri-snRNP assembly or in steps
following tri-snRNP assembly but prior to
spliceosome activation, since the U4-G58C mutation leads to
accumulation of the B complex (Hu et
al., 1995). Other mutations in U4 stem I predicted to disrupt
base pairing with U6 do not display
synthetic lethality with snu114 mutations. Further work is
required to understand the specific
sensitivity of only position U4-G58A in U4 stem I and the reason
for its genetic interactions with
SNU114.
Snu114p senses the state of U6-5’ splice site and U2/U6 base
pairing
In addition to sensing the state of U4/U6, we show that Snu114p
interacts genetically with
key positions in the U6-ACAGAGA that base pairs with the 5’
splice site. This suggests that
successive interactions of Snu114p with U4 and U6 may facilitate
the transitions that occur during
spliceosome activation. Notably, Prp8p also interacts with the
U6-ACAGAGA sequence: both
Prp8p and U6-A51 crosslink to the 5’ splice site before both
first and second steps of splicing (KIM
and ABELSON 1996; REYES et al. 1999; SONTHEIMER and STEITZ
1993). However, since prp8
mutations only partially suppress U6-A51 mutations which cause
5’ splice site misalignment and
result in defective exon ligation (COLLINS and GUTHRIE 1999),
Snu114p, together with Prp8p, may
coordinate U6 at the 5’ splice site. That snu114 alleles
interact genetically with mutations of U6-
G52 but not with U2-A25 suggests that the Snu114p genetic
interactions with U6-G52 occur during
U6-5’ splice site base pairing rather than during formation of a
tertiary interaction with U2 for the
second step of splicing (MADHANI and GUTHRIE 1994). Additional
mutations in U6 or the pre-
mRNA designed to stabilize the U6-5’ splice site interactions
combined with snu114 G-domain
mutations would provide further support for the hypothesis that
the G-domain senses U6-5’ splice
site interactions.
-
Frazer et. al. page 27
Brr2p dependent unwinding of U2/U6 base pairing during
spliceosome disassembly is also
regulated by the guanine nucleotide state of Snu114p (SMALL et
al. 2006), therefore, we
investigated genetic interactions between snu114 and various U2
and U6 snRNA mutations that
influence U2/U6 base pairing. Formation of U2/U6 helix I is
important for promoting the first and
second chemical steps of splicing (MADHANI and GUTHRIE 1992;
MEFFORD and STALEY 2009;
RYAN and ABELSON 2002) and unwinding of the U2/U6 helix I is
required for disassembly of the
spliceosome (SMALL et al. 2006). Synthetic lethality is only
observed between N-terminus and G
domain snu114 mutations in combination with mutations in U2 and
U6 that are predicted to disrupt
or stabilize U2/U6 helix I base pairing. The synthetic lethality
patterns observed between Snu114p
with U6 and U2 helix I mutations suggest that Snu114p senses the
state of U2/U6 base pairing
interactions. This hypothesis is supported by the suppression of
synthetic lethality by compensatory
mutations that restore base pairing in U2/U6 helix I. Therefore,
at least for the U2/U6 helix I it is
clear that synthetic lethality was solely due to the disruption
of base pairing between U2 and U6 in
this region. The lack of suppression of synthetic lethality by
compensatory mutations at other
positions predicted to restore base pairing does not preclude a
role for Snu114p in sensing base
pairing at these positions. Rather it suggests a possible
additional role, besides base pairing, for the
positions where synthetic lethality can not be suppressed by
compensatory mutations.
The specificity of genetic effects of U6 mutations to the U2/U6
conformation is confirmed
by the lack of genetic interactions between Snu114p and U4 in U4
stem I. Although positions in
U4 stem I which participate in the U4/U6 complex opposite
U6-A56,U57 were not assayed, three
other U4 stem I alleles displayed no synthetic lethality with
snu114 alleles. This suggests that
SNU114-U6 genetic interactions are related to U6 in the U2/U6
conformation rather than the U4/U6
conformation. That synthetically lethal effects are specific to
disruption of U2/U6 helix I is
supported by the lack of synthetic lethality with mutations and
deletions at U2-U24 and U2-A25
which form a non-base paired bulge in U2/U6 helix I.
-
Frazer et. al. page 28
Three differences are observed between U2 and U6 synthetically
lethal and compensatory
interactions. First, the snu114 N-terminus deletion mutation is
synthetically lethal with mutations
in U6 that disrupt base pairing whereas there is no synthetic
lethality of the N-terminus deletion
mutation with U2 mutations that disrupt base pairing. Second,
insertion mutations in opposite sides
of helix Ia display different genetic effects with SNU114. An
insertion mutation of U6 in helix Ia
(U6-Ins 1U A53/U54) is viable with all snu114 mutations tested.
On the other hand, an insertion
mutation of U2 in helix Ia (U2-Ins 1U U28/A29) displays
synthetic lethality with snu114 G domain
mutation V266P (G4). It is known that insertions at this
location in U6 inhibit the first step of
splicing whereas insertions at this location in U2 inhibit the
second step of splicing (MCGRAIL et al.
2006; MCPHEETERS and ABELSON 1992). Third, suppression of
synthetic lethality by compensatory
mutations was successful with mutations in U2 that restored base
pairing with U6 mutations in helix
Ia and Ib, however, mutations in U6 that restored base pairing
with U2 mutations in helix Ia and Ib
were not found to suppress synthetic lethality with snu114
mutations. Overall, these differences
suggest additional roles for nucleotides in U2 that form helix
Ia and Ib during splicing. These roles
could be manifested when U2 and U6 are not base paired either
before formation of U2/U6 helix I,
after formation of U2/U6 helix I or when U2/U6 helix I is
destabilized by Prp16p between the
catalytic steps of splicing (MEFFORD and STALEY 2009).
Snu114p interactions and orientation with U5 snRNA
As Snu114p is an integral protein of the U5 snRNP, we
investigated synthetic lethality
between snu114 and twenty-five U5 mutations. Mutations in U5
displayed synthetic lethality with
the snu114 N-terminal deletion mutation and G domain mutations.
U5 was the only snRNA to
show synthetic lethality with the snu114 C-terminal P860K (IVa)
mutation. The snu114 G domain
mutation V266P (G4) was synthetically lethal with U5 loop 1
mutations while the snu114 C-
terminal domain mutations G646R (III) and P860K (IVa) were
synthetically lethal with a U5 IL1
mutation. This suggests an orientation of Snu114p with U5 within
the spliceosome such that U5
-
Frazer et. al. page 29
links the G domain to the other domains of Snu114p. We observed
spatially distinct genetic
interactions between the C-terminus of Snu114p, which displayed
synthetic lethality with U5 IL1,
and the G domain which did not. This orientation suggests that
U5 and Snu114p may work
together to transmit information from the G domain to the
C-terminal domain which has been
shown to interact with other splicing factors (BRENNER and
GUTHRIE 2005; LIU et al. 2006). It is
possible that the state of the snRNAs sensed through the G
domain of Snu114p is transduced
through the C-terminal end of Snu114p, perhaps through a
structural change/domain shift, which is
the mechanism of function of other regulatory GTPases (JORGENSEN
et al. 2003).
The SNU114 N-terminal domain also interacts genetically with U5
IL1 which suggests that
it may also be involved in contacting U5 in this region along
with U5 loop 1. This is the first
evidence that suggests a conformation for the N-terminal domain
of Snu114p (Figure 8), which
cannot be modelled on EF-2. Whether the N-terminal domain
interacts with U5 loop 1 and U5 IL1
at the same or at different times is unknown. For both the N-
and C-terminal domains of Snu114p
to interact with U5 IL1 suggests a long range intramolecular
interaction such that the N-terminus
and domain IV come into close proximity with U5 IL1 if these
interactions are simultaneous. These
interactions may also involve Prp8p since it has been found to
crosslink to position C112 in U5 IL1
(DIX et al. 1998). The only other genetic interactions found
with the snu114 ΔN mutation were
with mutations in the U6 snRNA. The fact that no U2 or U4
synthetically lethal interactions were
found with the snu114 ΔN mutation suggests that the Snu114p
N-terminus may be involved in U5
and U6 interactions at the 5’ splice site.
We have identified an extensive genetic interaction network
between the spliceosomal
GTPase Snu114p and the snRNAs of the spliceosome (Figure 8). By
combining four new snu114
mutations with five previously known mutations we have
distinguished different roles for distinct
regions of Snu114p and propose an orientation for Snu114p with
U5 snRNA. It appears that
Snu114p recognizes the state of important RNA/RNA interactions
within the spliceosome (Figure
8). Snu114 G domain mutations exacerbate mutations in U4 that
change U4/U6 and U2/U6 base
-
Frazer et. al. page 30
pairing. Snu114 G domain mutations also exacerbate mutations in
U6 that increase base pairing
with the 5’ splice site. Mutations in the snu114 G domain and
N-terminal domain exacerbate
mutations in U5 at positions which align the exons in the
catalytic core. Thus the Snu114p G
domain may have a significant role in signalling the state of
the snRNAs throughout splicing
(Figure 8). Additionally, snu114 mutations in the N-terminal
domain and domains III and IV
exacerbate mutations in U5 internal loop 1 which could serve to
link the N- and C-terminal domains
of Snu114p functionally. These genetic interactions with U5 also
provide a structural link between
the G domain and C terminal domain, and therefore a link between
the proposed sensing and
transducing regions of Snu114p. Thus by analysis of the genetic
interactions between distinct
domains in Snu114p and the snRNAs of the spliceosome we have
defined a functional and spatial
network of interactions that we propose is essential for
spliceosome function. Further work is
required to test the new snu114 mutations with alleles in
proteins that have been found to interact
with Snu114p previously (BRENNER and GUTHRIE 2005). This work
would establish how the
genetic interactions we found between Snu114p and snRNAs fit
into the network of Snu114p-
protein interactions in the spliceosome and would provide an
insight into whether these genetic
interactions represent direct or indirect physical interactions
with Snu114p. Conversely, genetic
interactions between snRNA mutations and other splicing protein
alleles should be investigated to
discover how the Snu114p-snRNA interactions fit in with other
potential snRNA-protein networks.
-
Frazer et. al. page 31
SUPPLEMENTARY DATA
Supplementary data are available at the Genetics journal
online.
-
Frazer et. al. page 32
ACKNOWLEDGEMENTS
We thank Riz Alvi for constructing the U5KO, U2KO and U6KO
strains, Moses Tandiono for
constructing the YSNU114/U4KO strains and pRS416-Snu114-U4
plasmid and members of the
O’Keefe lab for critical reading and advice on the manuscript.
This work was funded by a grant to
RTO by the Wellcome Trust (079536).
-
Frazer et. al. page 33
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TABLE 1. Yeast strains used in this study
Strain Genotype Reference Y25023 MATa/α; his3Δ1/ his3Δ1;
leu2Δ0/leu2Δ0;
lys2Δ0/LYS2; MET15/met15Δ0; ura3Δ0/ura3Δ0;
YKL173w::kanMX4/YKL173w
EUROSCARF
YSNU114KO1 MATa; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0;
YKL173w::kanMX4; pRS416-Snu114
This study
YSNU114KO2 MATα; his3Δ1; leu2Δ0; ura3Δ0; YKL173w::kanMX4;
pRS416-Snu114
This study
YU5KO (YROK2) MATa; his3Δ200; leu2Δ1; trp1Δ63; ura3-52;
snr7::kanMX4; pRS416-U5
O’KEEFE, 2002
YU2KO MATa; snr20::kanMX4; pRS416-U2 This study YU6KO MATa;
his3Δ1; leu2Δ0; lys2Δ0; met15Δ0;
snr6::kanMX4; pRS416-U6 This study
YSNU114/U5KO MATα; his3Δ200; leu2Δ0; trp1Δ63; ura3Δ0;
YKL173w::kanMX4; snr7::kanMX4; pRS416-Snu114-U5
This study
YSNU114/U2KO MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0;
YKL173w::kanMX4; snr20::kanMX4; pRS416-Snu114-U2
This study
YSNU114/U6KO MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0;
YKL173w::kanMX4; snr6::kanMX4; pRS416-Snu114-U6
This study
YSNU114/U4KO1 MATα; his3Δ1; leu2Δ0; ura3Δ0; YKL173w::kanMX4;
snr14::hphNT1; pRS416-Snu114-U4
This study
YSNU114/U4KO2 MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0;
YKL173w::kanMX4; snr14::hphNT1; pRS416-Snu114-U4
This study
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Frazer et. al. page 40
TABLE 2. snu114 mutations used in this study snu114 mutation
Location Viability Temperature
sensitivitya Reference
ΔN Deletion of N-terminal domain Viable 40 BARTELS et al., 2002
K146I snu114-15, Domain I (G domain) Viable 37 BRENNER and GUTHRIE,
2005 P216N snu114-12, Domain I (G domain) Viable 37 BRENNER and
GUTHRIE, 2005 V235D Domain I, G domain Viable 40 This study L236D/P
Viable 40 This study L236E Viable This study V238D Viable 39 This
study V238E/P Viable This study C264D Viable 40 This study F265D
Lethal ND This study V266D/H/W Lethal ND This study V266A/Y Viable
This study V266F/K/S Viable 40 This study V266P Viable 39 This
study I312D Viable 40 This study I312E/P Viable This study
F313D/H/K/R/S Lethal ND This study F313A/E Viable 40 This study
F313Q Viable 39 This study F313T/W Viable This study L381P
snu114-14, Domain I (G” domain) Viable 39 BRENNER and GUTHRIE, 2005
V562D/K/S/W Domain II Lethal ND This study V562A Viable 40 This
study L563D/H/Y Lethal ND This study L563A/M Viable This study
L563F Viable 37 This study L563P Viable 39 This study L563K/S/W
Viable 40 This study V562L563K/S/W Lethal ND This study V562AL563A
Viable 40 This study G646R snu114-30, Domain III Viable 37 BRENNER
and GUTHRIE, 2005 M842R snu114-40, Domain IVa Viable 37 BRENNER and
GUTHRIE, 2005 P860D Domain IVa Viable 40 This study P860E Viable
This study P860K Viable 39 This study a Blank entry indicates that
the mutant was not temperature sensitive up to 40°; ND indicates
that temperature sensitivity was not assayed.
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Frazer et. al. page 41
TABLE 3. snRNA mutations used in this study Mutation Location
Reference U5 C79A Invariant positions of internal loop 1 – 5’ side
This study G80C This study G80U This study G80A This study ΔC79
This study ΔC79G80 This study ΔC79-A81 This study ΔA78-A81 This
study G113U Invariant positions of internal loop 1 – 3’ side This
study C112A This study C111U This study ΔC111 This study ΔC112G113
This study ΔA41-U74 Variable stem loop O’KEEFE et al., 1996 U5
short form O’KEEFE et al., 1996 A100G Loop 1 This study A100UC101G
This study ΔG93 O’KEEFE and NEWMAN, 1998 ΔC94C95 O’KEEFE and
NEWMAN, 1998 ΔU96U97 O’KEEFE and NEWMAN, 1998 ΔA100 O’KEEFE and
NEWMAN, 1998 ΔC101 O’KEEFE and NEWMAN, 1998 Ins 1U U97/U98 O’KEEFE
and NEWMAN, 1998 Ins 1U G93/C94 MCGRAIL et al., 2006 Ins 1U C94/C95
MCGRAIL et al., 2006 U2 G20A/U 5’ to helix Iba This study C22A/G
Helix Iba MADHANI and GUTHRIE, 1992 U23C/G MADHANI and GUTHRIE,
1992 U24A/C/G Bulge between helix Ia and Iba MADHANI and GUTHRIE,
1994 ΔU24 MADHANI and GUTHRIE, 1994 A25C/G/U MCPHEETERS and
ABELSON, 1992 ΔA25 MCPHEETERS and ABELSON, 1992 A27C, U28C Helix
Iaa MADHANI and GUTHRIE, 1992 Ins 1U U28/A29 MCPHEETERS and
ABELSON, 1992 Ins 1U A30/A31 3’ to helix Iaa MCGRAIL et al., 2006
A31U This study Ins 1U G32/U33 MCGRAIL et al., 2006 U4 C14G Stem
IIb LI and BROW, 1996 U57A Stem Ib This study G58A MADHANI et al.,
1990 C59U MADHANI et al., 1990 U60C MADHANI et al., 1990 U64G This
study U64C This study G65A This study U64C,G65A This study
A66UA67UA68G (cs-1)
3’ to stem I LI and BROW, 1996 LI and BROW, 1996
U6 A47G Base-paired with pre-mRNA MADHANI et al., 1990 C48A This
study A49C This study G50U 5’ to helix Iaa MADHANI et al., 1990
G52U MADHANI et al., 1990 A53U This study
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Frazer et. al. page 42
Ins 1U A53/U54 This study U54C Helix Iaa This study G55A This
study U54C,G55A This study Ins 1U G55/A56 MCGRAIL et al., 2006
A56C, U57C MADHANI and GUTHRIE, 1992 C58U MADHANI and GUTHRIE, 1992
A59C Helix Iba MADHANI and GUTHRIE, 1992 C61G MADHANI et al., 1990
A62C 3’ to helix Ib This study a U2:U6 complex; b U4:U6 complex
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Frazer et. al. page 43
TABLE 4. Summary of the genetic interactions of snu114 and U4
snRNA mutations snu114 mutations
U4 mutations
WT ΔN K146I (G1) (114-15)
P216N (G2) (114-12)
V266P (G4)
F313Q (G5)
L381P (G’’) (114-14)
L563F (II)
G646R (III) (114-30)
M842R (IVa) (114-40)
P860K (IVa)
WT + + + + + + + + + + + G14C + + + + + + + + + + + U57A + + + +
+ + + + + + + G58A + + - + +/- +/- + + +/- +/- + C59U + + + + + + +
+ + + + U60C + + + + + + + + + + + U64C + + +/- + + + + + + + +
U64G + + + + + + + + + + + G65A + + +/- + + + + + + + + U64C,G65A +
+ - + + + + + + + + A66U,A67U,A68G (U4-cs1)
+ + - +/- - - +/- + + + +
- indicates synthetically lethal interaction; +/- indicates
synthetically sick interaction, and + indicates WT growth after 3
days at 30°.
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Frazer et. al. page 44
TABLE 5. Summary of the genetic interactions of snu114 and U6
snRNA mutations snu114 mutations
U6 mutations
WT ΔN K146I (G1) (114-15)
P216N (G2) (114-12)
V266P (G4)
F313Q (G5)
L381P (G’’) (114-14)
L563F (II)
G646R (III)
(114-30)
M842R (IVa) (114-40)
P860K (IVa)
WT + + + + + + + + + + + A47G + + + + + - + +/- + +/- + G50U + +
+/- + - - + + + - + G52U + + +/- + - - + + + - + A53U + + - + - -
+/- + + + + Ins 1U A53/U54 + + + + + + + + + + + U54C + + - + + +/-
+ + + + + G55A + + - + - +/- + + + + + U54C,G55A + + - + - +/- + +
+ + + Ins 1U G55/A56 + + + + + + + + + + + A56C,U57C + +/- - +/- -
+/- +/- + + +/- + C58U + + + + +/- + + + + + + A59C + +/- +/- + - -
+ + + + + C61G + - - +/- - - - + + - + A62C + + + + + + + + + + + -
indicates synthetically lethal interaction, +/- indicates
synthetically sick interaction, and + indicates WT growth after 3
days at 30°.
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Frazer et. al. page 45
TABLE 6. Summary of the genetic interactions of snu114 and U2
snRNA mutations snu114 mutations
U2 mutations
WT ΔN K146I (G1) (114-15)
P216N (G2) (114-12)
V266P (G4)
F313Q (G5)
L381P (G’’) (114-14)
L563F (II)
G646R (III)
(114-30)
M842R (IVa) (114-40)
P860K (IVa)
WT + + + + + + + + + + + C22A + + + + - + + + + + + C22G + ND +
+ - + + + + + + U23C + + + + - +/- + + + + + U23G + ND + + - +/- +
+ + + + U24A + ND ND ND + + ND + ND + + U24C + ND ND ND + + ND + ND
+ + U24G + + + + + + + + + + + ΔU24 + + + + + + + + + + + A25C + ND
ND ND + + ND + ND + + A25G + + + + + + + + + + + A25U + ND ND ND +
+ ND + ND + + ΔA25 + + + + + + + + + + + A27C,U28C + + + + - +/-
+/- + + + + Ins 1U U28/A29 + + + + - + + + + + + A31U + + + + - + -
+ + + + Ins 1U A30/A31 + + + + + + + + + + + Ins 1U G32/U33 + + + +
+ + + + + + + - indicates synthetically lethal interaction; +/-
indicates synthetically sick interaction, and + indicates WT growth
after 3 days at 30°; ND indicates comparison was not done.
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Frazer et. al. page 46
TABLE 7. Summary of the genetic interactions of snu114 and U5
snRNA mutations
U5 mutations
snu114 mutations WT ΔN K146I
(G1) (114-15)
P216N (G2) (114-12)
V266P (G4)
F313Q (G5)
L381P (G’’) (114-14)
L563F (II)
G646R (III) (114-30)
M842R (IVa) (114-40)
P860K (IVa)
WT + + + + + + + + + + + C79A + + + + + + + + + + + G80C + ND ND
ND + + ND + ND + + G80U + + + + + + + + + + + G80A + ND ND ND + +
ND + ND + + ΔC79 + + + + + + + + + + + ΔC79,G80 + ND ND ND + + ND +
ND + + ΔC79-A81 + ND ND ND + + ND + ND +/- + ΔA78-A81 + + + + + + +
+ + +/- + C111U + + + + + + + + + + + ΔC111 + + + + + + + + + - +
C112A + + + + + + + + + + + G113U + + + + + + + + + + + ΔC112,G113
+ - + + + + + + - - - ΔA41-U74 + + + + + + + + + + + U5 short form
+ + + + + + + + + + + A100G + + + + + + + + + + + A100U,C101G + + +
+ - + + + + + + ΔA100 + + + + + + + + + + + ΔC101 + + + + + + + + +
+ + ΔG93 + + + + + + + + + + + ΔC94,C95 + - + + - + + + + + +
ΔU96,U97 + - + + - + + + + + + Ins 1U U97/U98 + + + + + + + + + + +
Ins 1U G93/C94 + + + + - + + + + + + Ins 1U C94/C95 + + + + + + + +
+ + + - indicates synthetically lethal interaction; +/- indicates
synthetically sick interaction, and + indicates WT growth after 3
days at 30°; ND indicates comparison was not done.
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Frazer et. al. page 47
FIGURE LEGENDS
FIGURE 1. Location of snu114 mutations. A. Linear diagram of
Snu114p based on sequence
homology to yeast elongation factor (Eft2p). Domains are
labelled in Roman numerals. The N-
terminal domain is unique to Snu114p. Within domain I, the G
domain, the GTPase conserved
motifs are labelled 1-5. The G domain also contains a G’’
element which is unique to Snu114p and
Eft2p. Ts snu114 mutations used to analyze genetic interactions
with snRNAs are displayed above
the linear diagram and mutations from a previous study (BRENNER
and GUTHRIE 2005) are shown
below the linear diagram. B. Three dimensional predicted
structure of Snu114p based on sequence
homology with Eft2p. The N-terminal domain (residues 1-121) and
a region from the C-terminus
(residues 982-1008) were omitted. Colors correspond to the
linear diagram of Snu114p. Yellow
spheres indicate location of amino acids mutated in this study
and red spheres indicate amino acids
mutated by Brenner and Guthrie (2005).
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Frazer et. al. page 48
FIGURE 2. Growth defects, protein expression levels and splicing
defects of snu114 mutants. A.
Growth of WT and snu114 strains. Serial dilutions of WT and
snu114 strains grown at 16° for 10
days or 25°, 30°, 37° and 39° for 3 days. snu114 mutant L563F
exhibits a growth defect at 37°
whereas the remaining snu114 mutants exhibit a growth defect at
39°. B. Protein expression levels
of Snu114p monitored at permissive (30°) and restrictive
temperatures (39°). Protein levels in WT
and snu114 strains remained constant after a shift to 39°
compared to the loading control, yeast
glucose 6-phosphate dehydrogenase (Zwf1p). C. snu114 mutations
inhibit pre-mRNA splicing.
RNA was extracted from WT or snu114 strains grown at the
restrictive temperature and analyzed
by primer extension of pre-U3 snoRNA. Pre-U3A, pre-U3B and
mature U3 snoRNA are indicated
to the right of the panel.
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Frazer et. al. page 49
FIGURE 3. Genetic interactions between snu114 and U4 snRNA
mutations. Strain
YSNU114/U4KO1 containing the WT SNU114 and U4 snRNA alleles on a
URA3 plasmid was
transformed with WT or snu114 and U4 snRNA alleles on separate
plasmids. Transformants were
transferred to 5FOA to evict the URA3 plasmid containing the
wild type genes and determine the
synthetic lethality between mutant alleles. The annotated
structure of base paired U4/U6 in the
conformation within the tri-snRNP is displayed above the panels.
Positions of substitutions are
underlined. The U4-cs1 mutation is A66U,A67U,A68G.
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Frazer et. al. page 50
FIGURE 4. Genetic interactions between snu114 and U6 snRNA
mutations. Strain
YSNU114/U6KO containing the WT SNU114 and U6 snRNA alleles on a
URA3 plasmid was
transformed with WT or snu114 and U6 snRNA alleles on separate
plas