Banting and Best Department of Medical
Research and Department of Molecular and Medical Genetics,
University of Toronto, Toronto, Ontario, Canada M5G 1L6
Received 8 September 1997/Returned for modification 16 October
1997/Accepted 20 January 1998
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INTRODUCTION |
Precursor-mRNA (pre-mRNA) splicing
takes place in the spliceosome through a two-step transesterification
reaction. At least 40 splicing factors have been identified by genetic
means in the yeast Saccharomyces cerevisiae. Most have been
implicated in specific steps of the splicing pathway (31,
41). During the process of spliceosome assembly, small nuclear
RNAs (snRNAs) and the pre-mRNA substrate, in association with protein
factors, undergo extensive conformational changes which establish
RNA-RNA interactions that are important for both splicing reactions.
Among these factors are the DExD or DExH proteins: RNA-dependent
ATPases (possibly RNA helicases), which include Prp2p, Prp5p, Prp16p,
Prp22p, Prp28p (26, 31), Prp43p (2), and Slt22p
(also called Brr2p) (22, 34, 53). Their functions are
essential for the formation and maintenance of RNA-RNA interactions in
the spliceosome.
With few exceptions (28, 29, 36), Watson-Crick base pairing
is important for most RNA-RNA interactions in the formation of the
spliceosome (26). In particular, intermolecular base-pairing interactions that occur between U2 and U6 snRNAs, forming helices I and
II (Fig. 1A), are likely
to be involved in bringing the 5' splice site (through a
U6-5'-splice-site interaction [21, 23, 42]) and the
branchpoint site (through a U2-branchpoint interaction
[37]) into proximity. This is necessary for the first-step reaction (Fig. 1A). Residues in both U2 and U6 snRNAs that
are important for either or both steps of splicing (11, 30)
form part of these interactions or lie nearby. This finding has led to
the suggestion that these RNA structures may form the so-called active
center for catalysis of the splicing reactions (27, 30). The
helix II portion of human U6 snRNA has also been shown to influence
dissociation of the U4-U6 snRNA duplex in vitro, perhaps by stabilizing
it (5).
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FIG. 1.
(A) Yeast U2 snRNA. The top diagram shows the structure
of yeast U2 snRNA with the 5'-end region, including stems I and II. BP
int, the region that interacts through base pairing with the
branchpoint (BP) site in pre-mRNA. The bottom diagram represents U2-U6
snRNA interactions (intermolecular helices Ia, Ib, and II), illustrated
in the context of BP-U2 (BP) and 5'-splice-site-U6 (5' SS)
interactions, and alignment of exons by U5 snRNA
(Alignment). Nucleotides involved directly in the
transesterification reaction are circled. Dots indicate the U2-U6 snRNA
helix II region that is mutated in the 11-nt substitution (Sub.)
mutation of U2 snRNA. The 11-nt substitution (12) used in
the genetic screen and other U2 snRNA mutations used to test allele
specificity are shown under U2 snRNA in the context of U2-U6 snRNA
interactions. (B) Growth phenotypes of yeast strains carrying the U2
snRNA mutations shown in panel A. (C) Yeast strain used in the genetic
screen. The strain (12) contains a chromosomal deletion of
the U2 snRNA gene (SNR20), which was replaced with
HIS3 (SNR20 ::HIS3), and
two plasmids carrying wt SNR20 (URA3 CEN-ARS,
i.e., a maintenance plasmid) and mutant snr20-11nt
(TRP1 CEN-ARS). Following EMS mutagenesis, cells harboring
extragenic mutants (slt's) that became synthetically lethal
with the mutant U2 snRNA were also sensitive to 5-FOA. (D) Growth
phenotypes of six slt mutants. In addition to conferring
synthetic lethality, these slt mutants all confer growth
defects at various elevated temperatures. Shown are the 2-day growth
phenotypes of the slt strains obtained following a series of
back-crosses with a wt strain (carrying SNR20) grown on
yeast extract-peptone-dextrose medium. Note that the slt15,
slt16, and slt22 strains all grow slowly at
30°C.
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The highly conserved loop 1 region of U5 snRNA is important for the
juxtaposition of the two exons, which is essential for the second step
of splicing (35). However, canonical base pairing is not
obviously involved in this interaction, suggesting that additional
factors are required for its establishment and maintenance. In fact,
two splicing factors, Prp8p (45, 47, 48) and Slu7p (6,
13), have been implicated in the alignment of the two exons
and/or in 3'-splice-site selection. In addition, a functional role for
U2 snRNA in the alignment of the two exons has been suggested by
site-specific cross-linking between the first nucleotide of the 3' exon
and the U23 and A30 nucleotides of U2 snRNA (33). Despite
the available information, it is not clear how different RNA structures
in the active center are coordinated, either spatially or temporally,
prior to and after the first splicing step.
The yeast U2 snRNA contains all conserved elements found in other
eukaryotic U2 snRNAs, despite its unusually large size (approximately 1,200 nucleotides [nt]) (Fig. 1A). The stem II region of U2 snRNA is
important for recognition of the branchpoint site and association of
the U2 snRNP with the pre-mRNA. Several factors, including Prp5p (a
DEAD-box protein), SF3a (Prp9p Prp11p Prp21p), and SF3b, have been
implicated in this function (40, 51, 54). In contrast, relatively little is known about the factors involved in subsequent steps of spliceosomal function during which the stem I region of U2
snRNA undergoes extensive conformational rearrangements to form the
U2-U6 snRNA interactions (see above and Fig. 1A).
We have devised a synthetic-lethality genetic screen to search
for such factors. In this screen, a mutant U2 snRNA carrying an
11-nt substitution in the stem I region (12) was used as the
starting mutation. Although this mutation can potentially perturb the
U2-U6 snRNA helix II interaction (Fig. 1A), it confers only a mild
growth defect, which is likely due to functional redundancy of the
helix Ib and helix II regions in U2-U6 snRNA (12). Our genetic screen yielded six slt (stands for synthetic
lethality with U2) mutants. We have characterized and cloned all the
SLT genes, two of which encode new splicing factors: Slt11p
(a possible RNA-binding protein), Slt22p (an RNA-dependent ATPase
[53]), and four previously characterized
factors, Slt15p (previously named Prp17p [Slt15p/Prp17p]),
Slt16p/Smd3p, Slt17p/Slu7p, and Slt21p/Prp8p. These factors are
required for either or both steps of splicing. Our genetic and
biochemical characterizations suggest that the functions of these
factors and of U2 snRNA may overlap that of invariant loop 1 of U5
snRNA in the alignment of the two exons in the spliceosome.
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MATERIALS AND METHODS |
Yeast strains and plasmids.
All yeast strains used in this
study were derived from W303-1A or -1B (MATa or
MAT
ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 can1-100).
The original strain used in the genetic screen, containing a
chromosomal deletion of SNR20 (the U2 snRNA gene), has been
described previously (12). A new SNR20
disruption was made by deleting the ClaI-HpaI
region of ~810 nt containing the 5'-end half of the U2 snRNA gene
(~750 nt) and by replacing it with the yeast HIS3 gene.
The wild-type (wt) SNR20 gene was carried on pRS316
(URA3 CEN-ARS). Two resultant haploids, YDX2299A (MATa) and YDX22100A (MAT), were used in
the subsequent genetic experiments. Chromosomal deletion of
SNR6 (the U6 snRNA gene) was constructed by deleting the
entire coding region and replacing it with the yeast HIS3
gene. Two haploid SNR6 strains, YXU91 (MAT)
and YXU92 (MATa), were obtained. Strains containing slt11, slt17, slt21, and
slt22 mutations in the SNR20 background were
crossed to both SNR6 strains in order to generate haploid strains carrying double deletions as well as slt mutations.
SNR20 and SNR6 were carried on the same
maintenance plasmid. These strains were used to test genetic
interaction among slt, U2, and U6 mutations. U6 snRNA
mutations (3-nt substitutions near the 3' end: designated a, b, c, and
d) have been described (12).
Strains containing slu mutations and U5 snRNA constructs
(U98A, U98C, and the U97C U99C double mutation [U97C/U99C]
[14]) and U5 snRNA constructs were provided by C. Guthrie (University of California, San Francisco). PCR was used to
subclone mutant U5 snRNA fragments into pRS315 (LEU2
CEN-ARS). The SNR7 disruption was constructed by
deleting the entire coding region and replacing it with the yeast
HIS3 gene. Two disruption strains, YXU37A
(MATa) and YXU37B (MAT), were used in
subsequent experiments. Strains containing chromosomal deletions of
both SNR20 and SNR7 were created by crossing the
two single-deletion haploid strains; a maintenance plasmid carrying
both the SNR20 and SNR7 genes was then introduced into the resultant heterozygous diploid before sporulation. The double-deletion strains (YXU53 and YXU54) were obtained from the progenies following sporulation and tetrad dissection. A strain containing the prp2-1 mutation and the PRP8 gene
were provided by J. Beggs (University of Edinburgh). prp2-1
and prp28-102 plasmids were obtained from R.-J. Lin (Beckman
Research Institute of the City of Hope) and T.-H. Chang (Ohio State
University), respectively. For the testing of synthetically lethal
interactions between slu (or prp) mutations and
U2 snRNA mutations, the slu (or prp) mutations were segregated genetically into the SNR20 strain through
at least two consecutive crosses with YDX2299A or YDX22100A. Following tetrad dissection, progeny haploids containing the slu (or
prp) mutation and SNR20 were selected and used
to test synthetic lethality with the U2 snRNA mutations. In order to
exclude differences in genetic background, at least four independent
SNR20 slu (or prp) isolates were used in these
experiments. Genomic fragments containing the prp2-1 and
prp28-102 mutations were introduced into W303-1A by two-step
gene replacement. Both mutations were then segregated to the
SNR20 background as described above.
EMS mutagenesis, screening, and genetic characterization.
In
the initial genetic screen, a yeast strain with a chromosomal deletion
of SNR20 (12) containing SNR20 and
snr20-11nt carried on URA3 and TRP1
plasmids, respectively, was subject to ethyl methanesulfonate (EMS)
mutagenesis to yield a survival rate of 10 to 20%. The surviving cells
were screened for sensitivity to 5-fluoroorotic acid (5-FOA) at 30°C.
This indicates dependence of viability on SNR20 (carried on
a URA3 plasmid), which reflects the lethality generated by
snr20-11nt in combination with extragenic mutations (i.e.,
synthetic lethality) (Fig. 1C). In order to eliminate artifacts (e.g.,
mutations which affect the uracil pathway), the snr20-11nt/TRP1 plasmids in 5-FOA-sensitive cells were
replaced by another SNR20 plasmid. The resultant cells that
were sensitive to 5-FOA were discarded. A scheme for genetic
characterization was designed to ensure that the slt mutants
were phenotypically and genetically appropriate (they should have
arisen from mutation at a single locus which also confers a recessive
growth defect). Briefly, the temperature-sensitive phenotype of each
original slt strain was first segregated into the
chromosomal SNR20 background by crossing the slt
strain with the wt strain (W303-1A). The resulting temperature-sensitive haploid was then back-crossed with the wt strain
at least three times to ensure that the temperature sensitivity phenotype was due to mutation at a single locus. The final
temperature-sensitive haploid was crossed with the newly constructed
SNR20 deletion strain (YDX2299A or YDX22100A). Synthetic
lethality was then tested in strains containing both the temperature
sensitivity mutation and SNR20 derived from the
above-described cross. These strains were also used to generate the
slt SNR20 plus SNR6 strains
described above.
Splicing extract preparation and in vitro splicing.
Yeast
whole-cell extracts were prepared from wt and slt cells
according to the method of Lin et al. (24) with
modifications. The substrate
32P-labeled yeast pre-actin
RNAwas synthesized by runoff transcription with T7 RNA polymerase and
32P[UTP] as described previously (24).
For in vitro splicing assays, equal volumes (10 µl) of whole-cell
extract and buffer component, containing labeled substrate, were mixed
and incubated at the temperatures indicated in the figures. Splicing
intermediates and products were resolved in a 5% polyacrylamide
gel.
RNA isolation and primer extension.
All slt
mutant strains used for RNA analysis contained SNR20 on the
chromosome (see above). Total yeast RNA isolation and primer extension
were performed as described previously (17).
Cloning by complementation.
In order to clone the wt
SLT genes, a YCp50-borne yeast genomic DNA library, CENBANK
A (38), was introduced into slt cells and the
transformants were selected for temperature resistance at 37°C.
Plasmids containing complementing genomic DNA fragments were recovered
from the positives. A minilibrary approach was used to define the
minimal complementing regions. The original complementing plasmid was
first digested with a variety of restriction enzymes, and the resulting
fragments were ligated to another vector with a different selective
marker. Total plasmid DNA prepared from these mini-libraries was
introduced into the slt strains, followed by selection for
full complementation. The overlapping region in plasmids rescued from
different mini-libraries contain slt-complementing open
reading frames. Nucleotide sequences of both ends of these fragments
were determined and then used to search the Saccharomyces
Genome Database (http://genome-www.stanford.edu/Saccharomyces/) for
a match. DNA sequences of complementing regions were retrieved, and
fragments containing only single open reading frames were tested for
complementation of both growth defect and synthetic lethality.
Genetic analysis of synthetic lethality.
In order to examine
potential synthetic lethality of two mutations, a heterozygous diploid
was first generated from two parental slt (slu or
prp) haploid strains and was then subjected to sporulation and tetrad dissection. If haploids containing both mutations are viable, three types of tetrads should be obtained: parental ditype (PD;
each spore inherits either of the original mutations), nonparental ditype (NPD; two spores inherit both mutations, while the other two
inherit neither mutation, i.e., it has the wt phenotype), and tetratype
(T; two spores inherit either of the original mutations, one inheriting
both mutations and the remaining one inheriting neither mutation, i.e.,
it has the wt phenotype). However, if the combination of both mutations
is lethal, only PD tetrads can be obtained from such a diploid. T
tetrads should contain only three viable spores (one wt spore and two
spores with either mutation), and NPD tetrads should contain only two
wt spores; the lack of complete (i.e., with four viable spores) NPD and
T tetrads indicates that the two mutations in question are
synthetically lethal. In most cases, at least 20 scoreable tetrads from
each heterozygous diploid were analyzed phenotypically to determine
synthetic lethality.
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RESULTS |
Genetic screening, characterization, and molecular cloning of
SLT genes.
The U2 snRNA mutation
(snr20-11nt) used in the search for synthetic-lethality
mutants contains an 11-nt substitution in the stem I region (Fig. 1A)
(12). Although such a substitution may potentially disrupt
the proposed U2 snRNA stem structure and perturb the helix II
interaction of U2-U6 snRNA, it confers only a mild growth defect at
both 37 and 16°C (12) (Fig. 1B). Cells containing the wt
U2 snRNA gene (SNR20) carried on a
URA3-marked plasmid and snr20-11nt on a
TRP1-marked plasmid were mutagenized with EMS. Surviving
cells were screened for extragenic mutations that failed to allow
growth at 30°C in the absence of SNR20 (as indicated by
sensitivity to 5-FOA). These extragenic mutations result from lethality
generated by snr20-11nt in combination with a second mutation (Fig. 1C), i.e., they produce synthetic lethality, which suggests functional interaction between the two gene products. These
mutations were designated slt mutations (for synthetic
lethality with U2 snRNA).
On the basis of 5-FOA sensitivity, nine candidate slt mutant
strains were isolated from approximately 8,000 colonies that survived
EMS mutagenesis. In the presence of SNR20, all mutant strains displayed a temperature-sensitive growth defect (see below). They were crossed with prp strains in our collection to test
for complementation. slt21 failed to complement
prp8-1 at 37°C, indicating that it might correspond to a
new allele of PRP8. The complementation test involving
slt15 and prp17-1 yielded an ambiguous result. However, following the cloning of SLT15 gene, it became
clear that slt15 is allelic to prp17-1. Each
slt mutation occurs at a single locus and confers a
recessive growth defect (Fig. 1D and see Materials and Methods).
The splicing defect associated with five slt mutations,
slt11, -15, -16, -17, and
-22 (Fig. 1D), representing five different genes, was
determined. In vitro splicing assays were performed with extracts
prepared from wt, slt11, slt22, and
slt17 cells. At 25°C the splicing activities of
slt11 and slt22 extracts were lower than that of
the wt extract (Fig. 2A, lanes 2 and 3), and at 33°C neither extract
had detectable splicing activity (Fig. 2A, lanes 5 and 6). No
preferential accumulation of intermediates was observed, suggesting
that both mutations affect pre-mRNA splicing prior to or at the first
step. A first-step splicing defect was also observed in vivo for both
mutations (data not shown). When the slt17 extract was
assayed at 25 or 33°C, reduced splicing activity was detected (Fig.
2A, lanes 11 to 14). Accumulation of splicing intermediates was
observed following a 20-min incubation at either temperature (Fig. 2A,
lanes 12 and 14), suggesting that the slt17 mutation affects
primarily the efficiency of the second-step reaction. This observation
is consistent with molecular cloning data which show that
slt17 is a new allele of SLU7.
In vivo splicing defects in slt15 and slt16
mutants were detected by primer-extension assays. Total yeast RNA was
isolated from these mutant cells before and following a shift from
25°C to a nonpermissive temperature (37°C). The levels of spliced
and unspliced actin and U3 RNAs were measured by primer extension with
labeled oligonucleotides complementary to the second exons of each
transcript. Inhibition of pre-mRNA splicing at 25°C was noted for
both mutant strains (Fig. 2B, lanes 2 and
12). This inhibition is consistent with the slow growth conferred at 25 and 30°C by both mutations (Fig. 1D and data not shown). The level of
mature mRNA was reduced in slt15 cells following the shift (Fig. 2B, lanes 6 to 8), while accumulation of unspliced precursors was
observed in slt16 cells at 37°C (Fig. 2B, lanes 13 and
14). These data indicate that both slt15 and
slt16 mutations affect pre-mRNA splicing in vivo.
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FIG. 2.
Splicing defects associated with slt
mutations. (A) In vitro splicing defects associated with the
slt11, slt22, and slt17 mutations.
Splicing reactions with 32P-labeled actin pre-mRNA
substrate were performed at 25 and 33°C for 20 min with whole-cell
extracts prepared from wt, slt11, slt22, and
slt17 cells. Precursor, intermediates (free 5' exon and
lariat intron-3' exon), and final products (5' exon-3' exon and
lariat intron) of the splicing reaction are indicated between the gels.
Arrows in lanes 12 and 14 indicate preferential accumulation of
lariat-intron-3'-exon intermediate in slt17 extract. (B)
Primer-extension analyses of inhibition of splicing in vivo. The left
gels show reduced levels of mature actin RNA in slt15 cells.
The right gels show inhibition of pre-U3 splicing in slt16
cells. The two upper bands labeled A and B correspond to pre-U3A and
pre-U3B, respectively. Cells were grown at 25°C for several
generations and then were shifted to 37°C. Total yeast RNA was
isolated before (grown at 25°C) and following a shift to 37°C for
the times indicated. Levels of spliced and unspliced RNAs were measured
by primer extension with labeled oligonucleotide complementary to the
second exon. Precursor and mature RNAs are indicated.
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The wt genes of slt11, -15, -16,
-17, and -22 were cloned by complementation with
a low-copy-number yeast genomic library. The minimum complementing
fragments for each SLT gene were identified by a
mini-library method (see Materials and Methods). Table
1 summarizes the results of molecular
cloning of SLT genes. Among the six SLT genes
identified in our genetic screen, two (SLT11 and
SLT22) encode new splicing factors and the remainder
correspond to splicing genes isolated previously.
Characterization of the slt22-1 mutation and the
RNA-dependent ATPase activity associated with Slt22p have been reported
(53). The interaction between Slt21p/Prp8p and U2 snRNA
and the role of Slt21p/Prp8p in formation of the active spliceosome
will be reported elsewhere.
SLT11.
SLT11, a new splicing-related gene,
corresponds to YBR065c. It encodes a 41-kDa protein of 364 amino acids. Two putative zinc fingers
(CX2CX17CX2C and
CX2CX6CX2C) are present in the
N-terminal region, while the central region (amino acids 151 to 299)
shows homology (27% identity and 40% similarity) to yeast ribosomal protein L25. The C terminus (approximately 30 amino acids) of Slt11p is
highly charged due to the presence of blocks of lysine residues. The
original slt11-1 mutation blocks splicing prior to the first
step (Fig. 2A, lane 5) without an apparent effect on spliceosome
assembly (unpublished data), suggesting that Slt11p exerts its function
immediately prior to the first-step splicing reaction.
SLT15/SLU4/PRP17.
SLT15/SLU4/PRP17 was first
isolated in a genetic screen for temperature sensitivity mutations that
also affect pre-mRNA splicing (prp17 [49])
and in another screen independently (slu4
[14]). The encoded protein contains WD repeats, which
are also present in another splicing factor, Prp4p (4, 17).
Prp17p is involved exclusively in the second step of splicing
(20). The new allele is named prp17-100. The
primary defect of the slt15/prp17-100 mutation was
associated with the reduction of mature RNA, and no preferential
accumulation of unspliced precursor was observed (Fig. 2B, lanes 6 through 8), suggesting that the mutation affects the stability of
mature RNA and/or, more likely, the second step of splicing.
SLT17/SLU7.
SLT17/SLU7 encodes a second-step
splicing factor (6, 13, 20) (Fig. 2A, lanes 11 through 14).
The gene product contains a so-called zinc knuckle that is similar to
one in retroviral nucleocapsid protein and that is required for
RNA-protein interaction (13). The slt17 allele is
renamed slu7-100. Both slu4-1/prp17-2 and
slu7-1 were isolated in a genetic screen for splicing
mutations that are synthetically lethal with mutations in invariant
loop 1 of U5 snRNA (14), a region that has been
implicated in alignment of the two exons in the second step of splicing
(35). However, Slu7p and Prp17p exert their functions at
different steps, with respect to that of hydrolysis of ATP by Prp16p
(20), a putative RNA helicase, whose function is required
for the second step (7, 8, 43).
SLT16/SMD3.
SLT16/SMD3 encodes a yeast core Sm
protein with homology to human SmD3 (39). slt16
is the first reported temperature-sensitive allele of this gene and is
renamed smd3-1. The protein is associated with all five
spliceosomal snRNAs and is thought to be involved in the biogenesis
of snRNPs. In an extract depleted of Smd3p, splicing is blocked
before or at the first step (39). A similar in vivo
splicing defect was observed for the slt16/smd3-1 mutation (Fig. 2B, lanes 12 through 14). The human SmD3 protein has been shown
to be cross-linked to the 5' splice site in a site-specific manner
(25, 52). To our knowledge, this study is the first time
that an Sm protein has been shown genetically to be important for the
function of a particular snRNA in splicing. However, it remains to
be determined whether this genetic interaction between Smd3p and U2
snRNA is direct (affects the function of U2 snRNA) or indirect
(affects the stability of snRNAs).
Genetic interactions of slt, slu, and
prp mutations with U2 snRNA.
We determined whether
the phenotypes of the slt mutations are specific for
the 11-nt substitution in the stem I region of U2 snRNA, with
which they were originally identified. We included in our genetic
analysis three sets of U2 snRNA mutations that lie in regions
that are involved in U2-U6 snRNA helices: helix Ia (G26A
and A27C), helix Ib (substitutions at G21), and helix II (11- and 9-nt
substitutions) (Fig. 1A). In addition to the six slt
mutations identified in this study, four slu (14)
and four prp mutations were also included (Table
2).
Three kinds of genetic interactions were observed between
slt and U2 snRNA mutations (Table 2): (i)
slt11-1 and slt15/prp17-100 showed virtually no
allele specificity, as they were synthetically lethal with all the U2
snRNA substitutions tested (with the exception of A27C for
slt11-1); (ii) slt16/smd3-1 and
slt17/slu7-100 showed synthetic lethality with specific
substitutions in the U2 snRNA part of the U2-U6 helix Ia and Ib
regions and with the helix II region; and (iii)
slt21/prp8-21 and slt22-1 were allele specific and lethal only with G21C in helix Ib and with the 11- and 9-nt substitutions in helix II but not with other U2 snRNA mutations tested.
Among the slu mutations, slu2 and slu3
showed no synthetic lethality with any of the U2 snRNA mutations
tested (Table 2). slu4/prp17-2 had the same allele
specificity as slt15/prp17-100. slu5 was lethal only with
two U2 snRNA mutations, G21C and G26A, but not with the 11-nt
substitution used in the genetic screen for slt mutants.
slu7-1 showed broader allele specificity than slt17/slu7-100 in that it was lethal with both G21A and G21U
in addition to G21C, which may affect the U2-U6 snRNA helix Ib
interaction.
The four prp mutations correspond to three RNA-dependent
ATPases involved in events which occur after the formation of
prespliceosome (Prp28p), concomitantly with U2-U6 snRNA
interactions (Prp2p), or in the second step (Prp16p). With the
exception of only one prp16-1 interaction, none of these
mutations showed synthetic lethality (Table 2). These results provided
genetic evidence that the RNA conformational rearrangements that are
affected by the original U2 snRNA mutation (11-nt substitution) is
specific for Slt22p (53).
Thus, the synthetically lethal interactions of most slt and
slu mutants are rather general with respect to mutations in
U2 snRNA. This may reflect the possibility that a large portion of the U2 snRNA and/or the U2-U6 snRNA structure, rather than
individual structural elements, is involved in interactions with these
factors.
Genetic interactions of slt mutations with U6 mutations
in U2-U6 snRNA helix II.
The original 11-nt substitution in U2
snRNA used in our genetic screen has the potential to perturb U2-U6
snRNA helix II (12). Consequently, we determined whether
this genetic interaction extended to nucleotides on the U6 snRNA
side of helix II. Yeast strains which contain slt mutations
in combination with double deletions of SNR20 and
SNR6 were constructed (Fig.
3A). These were used to determine whether
specific U6 snRNA mutations (12) were synthetically lethal with the slt mutations and whether partial
restoration of a mutationally disrupted helix II could suppress the
synthetic lethality that is observed with the original slt
mutations. (Note that we were unable to determine whether a
corresponding 9-nt U6 snRNA substitution conferred synthetic
lethality, since that mutation is lethal [12].) Two
slt mutations that encoded new splicing factors
(slt11-1 and slt22-1) and two that encoded
existing ones (slt17/slu7-100 and slt21/prp8-21)
were tested.
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FIG. 3.
Genetic interactions between slt mutations
and the U2-U6 snRNA helix II. (A) Representative yeast strain used
in the genetic tests. (B) U2 and U6 snRNA mutations used in the
genetic tests. The U6 snRNA mutations and their growth phenotypes
in a wt (SLT) background are described in reference
12. (C) Growth for 2 days at 30°C of four
slt strains containing various combinations of U2 and U6
snRNA mutations. Mutant U2 snRNA (TRP1-marked) and
U6 snRNA (LEU2-marked) plasmids were introduced into the
yeast strains shown in panel A. The resultant transformants were grown
on 5-FOA-containing selective medium. Note that the slt22-1
mutation confers slow growth at 30°C.
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slt22-1 showed synthetic lethality with three of the U6
snRNA mutations in the helix II region (U6-b, -c, and -d) (Fig.
3C). This is consistent with the observation that the RNA-dependent ATPase activity of this factor is related to U2-U6 snRNA helix II
and that a disrupted helix II (because of the U2 snRNA 11-nt substitution) is not an efficient in vitro substrate for Slt22p (53). slt11-1 showed similar synthetic lethality,
which at the current state of knowledge of this splicing factor we are
not yet able to explain. None of these synthetically lethal
interactions was suppressed by partial restoration of helix II through
the inclusion of the U2 snRNA 9- or 11-nt mutations (Fig. 3C). For slt22-1, this lack of suppression suggests that the helix II
structures formed by the U6-b, -c, and -d mutations are not efficient
substrates for the mutant protein.
Neither slt17/slu7-100 nor slt21/prp8-21 was
synthetically lethal with any of the U6 snRNA mutations tested
(Fig. 3C). This result suggests that these two factors are unlikely to
be involved in interactions with U2-U6 snRNA helix II but that they
are specific to interaction with U2 snRNA.
Genetic interactions among slt and slu
mutations.
The fact that the slt mutations are all
synthetically lethal with certain U2 snRNA mutations suggested that
at least some of them might form a functional unit(s). We assessed this
possibility by testing the synthetic lethality of pairs of
slt mutations, as well as of selected prp
mutations, by attempting to create double-mutant haploids (Table
3 and Materials and Methods). Failure to
obtain such haploids indicates that the two mutations in question are
synthetically lethal. The results of these experiments are summarized
in Tables 3 and 4 and Fig.
4.
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|
FIG. 4.
Summary of genetic interactions among factors involved
in either or both steps of splicing. Thick lines indicate synthetic
lethality at all temperatures. Thin lines indicate partial synthetic
lethality (Table 4). None of the slt16/smd3-1,
slt22-1, and prp2-1 mutations show synthetic
lethality with other mutations tested. Genetic interactions between
second-step mutations, including prp18 and prp8,
have also been described elsewhere (14, 20, 48). Pi,
inorganic phosphate.
|
|
Pairwise synthetic lethality was observed in strains
carrying the following mutations: slt11-1
(first-step mutation), slt15/prp17-100, slu4/prp17-2, slt17/slu7-100, slu7-1,
and prp16-1 (all second-step mutations). Two members of this
group, slt17/slu7-100 and prp16-1, also showed
weak but significant synthetic lethality with slt21/prp8-21 (the gene product is required for both splicing steps). Although not
identified in our screen, prp16-1 is also synthetically
lethal with one particular mutation, G26A, in U2 snRNA (Table 2)
and with four slt mutations (Table 4; Fig. 4). All other
combinations showed no significant synthetic lethality.
We suggest that Slt11p, Prp17p, Slu7p, Prp16p, and Prp8p form two
overlapping functional units. The fact that this group of factors
affects both splicing steps might indicate hitherto-unsuspected connections between the two reactions of pre-mRNA splicing.
Genetic interactions among slt and U5 snRNA
mutations.
All of our slt mutations were isolated as
synthetically lethal with a U2 snRNA mutation, yet two of them
(slt15 and slt17) are in the same genes
(slu4 and slu7, respectively) as mutations that
were identified originally as being synthetically lethal with U5
snRNA mutations (14). Furthermore, three (possibly four) Slt factors may act in functional groups (see above), which suggests the possibility of an interaction involving some of the Slt factors and
U5 snRNA.
This possibility was tested by constructing strains in which each of
five slt mutations, slt11-1,
slt15/prp17-100, slt17/slu7-100, slt21/prp8-21, and slt22-1, were segregated to a
SNR7 (SNR7 the U5 snRNA gene) background.
A plasmid carrying either of two U5 snRNA mutations, U98A and the
U97C/U99C double mutation, was then introduced into these strains to
test by plasmid shuffling for synthetic lethality (Fig.
5A).
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FIG. 5.
Synthetic lethality of slt mutants with loop
1 mutations of U5 snRNA. (A) Yeast strains carrying slt
mutations and a chromosomal deletion of the U5 snRNA gene
(SNR7::HIS3), with SNR7
on a URA3 CEN-ARS plasmid, were transformed with wt and
mutant U5 snRNA plasmids (LEU2 CEN-ARS). (B) Results of
5-day growth at 30°C of the resultant transformants on medium
containing 5-FOA. Note that slt22-1 and U5 snRNA double
mutants grew significantly slower than strains carrying either mutation
alone.
|
|
slt11-1 and slt21/prp8-21 showed synthetic
lethality at 30°C with both U5 snRNA mutations (Fig. 5B),
although slt21/prp8-21 showed some growth with the U5
U98A mutation at 25°C (Fig. 5B and data not shown). Weak synthetic
lethality was observed between slt22-1 and both U5
mutations at 25 and 30°C (Fig. 5B and data not shown). It has been
reported that the original slu4/prp17-2 and
slu7-1 mutations are synthetically lethal with the U5
snRNA U98A mutation (14); we found that
slt15/prp17-100 is lethal with both the U98A mutation and
the U97C/U99C double mutations and that slt17/slu7-100 is
lethal with U97C/U99C (data not shown).
Thus, four slt mutations that were identified on the basis
of synthetic lethality with U2 snRNA mutations are also
synthetically lethal with mutations in U5 snRNA loop 1. Two of
these, slt11-1 and slt21/prp8-21, affect the
first splicing step, while the tethering function of U5 snRNA is
required only for the second step (35). We conclude that
these factors, including the new one, Slt11p, may be involved in the
interactions involving both U2 and U5 snRNAs and in both splicing
steps.
Genetic interactions between U2 and U5 snRNAs.
The data
presented above indicate that members of a group of splicing factors,
which were identified originally on the basis of their genetic
interaction with U2 snRNA, also interact with one another and with
U5 snRNA. It is possible that these factors act together in
functions related to both U2 and U5 snRNAs, such as coordination of
the two steps of splicing and/or tethering of the two exons following
the first splicing step (35). This suggests that there may
be genetic interactions between U2 and U5 snRNAs. The two U5
snRNA mutations used in the genetic screen for slu
mutants (14), U98A/U99C, and a series of U2 snRNA
mutations were tested for genetic interaction with the yeast strain
shown in Fig. 6A.
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FIG. 6.
Genetic interactions (synthetic lethality and
suppression) between substitutions at the G21 position in U2 snRNA
and loop 1 mutations of U5 snRNA. (A) Yeast strain containing both
SNR20::HIS3 and
SNR7::HIS3. Mutant U2 snRNA
(TRP1 CEN-ARS) and U5 snRNA (LEU2 CEN-ARS)
plasmids were introduced into this strain to test for genetic
interactions. The resultant transformants were first grown on
5-FOA-containing medium at both 25 and 30°C. Cells containing
snr20-G21C and snr7-U97C/U99C failed to grow on
this medium; i.e., they are synthetically lethal. Other 5-FOA resistant
cells were then grown on selective medium at the temperatures
indicated. (B) Four-day growth at 25 and 30°C of U5 snRNA-wt,
-U98A, -U97C/U99C in combination with U2 snRNA-wt, -G21A, -G21C,
and -G21U in the absence of a maintenance plasmid. Note that U2-G21C is
synthetically lethal with U5-U97C/U99C. (C) Summary of genetic
interactions between U2 and U5 snRNAs and the locations of U2 and
U5 snRNA mutations in the context of other demonstrated RNA-RNA
interactions in the spliceosome. Positions 97, 98, and 99 in U5
snRNA loop 1 are enclosed in filled squares. Filled circles
indicate mutations at positions in U2 snRNA that are synthetically
lethal with the U5 snRNA mutations tested (see Table 5 for a
summary). G21 of U2 snRNA is enclosed in a filled square; mutations
at this position were able to suppress the U5 snRNA mutations
tested. The U2 snRNA part of the U2-U6 snRNA helix II is
shaded. Substitutions in this region (i.e., the 11-nt substitution)
cause synthetic lethality with slt mutations but not with U5
snRNA mutations. Lines from U23 and A30 in U2 snRNA to exon 2 indicate site-specific cross-linking (33).
|
|
In the helix II region, the original 11- or 9-nt substitutions of
U2 snRNA showed no synthetic lethality with either U5 snRNA mutation (Table 5), suggesting that U2-U6
snRNA helix II may not be involved directly in interaction
with U5 snRNA.
However, all substitutions at C22 and U23 of U2 snRNA in the helix
Ib region of U2-U6 snRNA were synthetically lethal or debilitating with either of the U5 snRNA mutations tested, even though these U2
snRNA mutations themselves conferred little or no growth defect at
these temperatures (Table 5). It is noteworthy that U23 is in close
contact with the first nucleotide of the 3' exon, which is tethered to
the 5' exon through interactions with loop 1 of U5 snRNA
(33).
Mutations at the adjacent G21 position of U2 snRNA showed a mixed
phenotype (Fig. 6B). At 25°C all three substitutions at G21
suppressed the slow growth conferred by the U5 U98A mutation, at 33°C
all three were synthetically lethal, and at 30°C (the intermediate
temperature) G21A and G21U had little effect while C21C was
synthetically lethal. With the U5 U97C/U99C double mutation, on the
other hand, only the G21A substitution suppressed at 25 and 30°C
while the G21C substitution was synthetically lethal at both
temperatures (Table 5; Fig. 6B).
G26A and A27C, the only two viable substitutions in the U2 part of
U2-U6 snRNA helix Ia (11, 30), were synthetically lethal with either U5 snRNA mutation tested (Table 5). No synthetic lethality was observed with mutations at other positions in U2 snRNA (e.g., substitutions G20C, U19G, C14G, and C14A [unpublished data]).
These results suggest an interaction of U5 snRNA loop 1 with the U2
portion of U2-U6 snRNAs helix I (Fig. 6B) but not with helix II.
While there is no biochemical evidence for a direct RNA-RNA interaction
between loop 1 of U5 snRNA and nt 21, 22, and 23 of U2 snRNA,
the genetic results presented here (Fig. 5B and Table 5) suggest that
an interaction may nevertheless exist. Given the role of U5 snRNA
loop 1 in alignment of the two exons (35), it may be that
the helix I region of U2 snRNA also plays a role in this function.
In support of this suggestion, we note that cross-linking experiments
(33) indicate that the helix I region of U2 snRNA is in
close contact with exon 2. This idea is consistent with the finding
that Slt17p/Slu7p and Slt2p/Prp8p are involved in 3'-splice-site
selection (6, 13, 20, 45, 47, 48).
| |
DISCUSSION |
The genetic screen described here has identified six yeast
splicing factors (called slt factors). Mutations in genes
encoding these factors become synthetically lethal with mutations in
the stem I region of U2 snRNA (corresponding to helix II of the
U2-U6 snRNA composite structure). Each of these splicing factors
affects either or both steps of splicing. One of these factors, Slt22p, is likely to be involved with U2-U6 snRNA helix II (53).
Three factors, Slt15p/Prp17p, Slt17p/Slu7p, and Slt21p/Prp8p, are
linked to the tethering of U5 snRNA loop 1 (14, 45). On
the basis of genetic interactions among some members of this group of
factors and other prp mutations, we suggest that Slt11p,
Prp17p, Slu7p, Prp16p, and Prp8p form two overlapping functional units.
Four slt mutations in this group are also synthetically
lethal with mutations in U5 snRNA loop 1. From this we conclude
that these factors, including a new one, Slt11p, may be involved in the
functions of both U2 and U5 snRNAs, perhaps in alignment of the two
exons in the yeast spliceosome and/or coordination of the two steps of
splicing. Genetic tests indicated an interaction between mutations in
loop 1 of U5 snRNA and those near the 5' end of U2 snRNA. Given the role of U5 snRNA loop 1 in the tethering of exons
(35), it may be that U2 snRNA also plays a role in this
function. Our findings suggest a mechanism for coupling and
coordination of the two steps of splicing.
The functions of U2-U6 snRNA helix I and helix II regions.
During maturation of the spliceosome, snRNAs and the pre-mRNA
substrate undergo conformational changes in order to juxtapose the
splice sites and to form structures that are important for the
subsequent steps of splicing (26). For both helix I and helix II of U2-U6 snRNA, there is evidence that the U6 snRNA
components may have functions that are independent of their interaction
in the helical structures. For example, mutations in the U6 snRNA components of yeast helix II are lethal and cannot be suppressed by
base-compensatory mutations in the corresponding U2 snRNA portion of U2-U6 snRNA (12). Similar genetic asymmetry was also
observed for nucleotides involved in the helix Ib interaction (11,
27, 28). Indeed, the 5' end of U2 snRNA itself may be
important for the second step of splicing. The genetic interactions of
both Slt17p/Slu7p and Slt21p/Prp8p with U2 snRNA are specific to
the U2 portion of U2-U6 snRNA helix II (Fig. 3). This observation differentiates the function of the 5'-end region of U2 snRNA from its role in U2-U6 snRNA helix II.
There is evidence that the role of U2-U6 snRNA helix II may be
exerted prior to the splicing reactions. In vitro dissociation of the
human U4-U6 snRNA duplex appears to be regulated by two elements in U6 snRNA: the center region (upstream of U4-U6
snRNA stem I, including the conserved ACAGAG motif that
recognizes the 5' splice site) and the 3'-end region, which forms U2-U6
snRNA helix II (5). It has been suggested that U2-U6
snRNA helix II may stabilize the U4-U6 snRNA duplex until the
spliceosome is fully assembled (5). It has also been
suggested that the energy released from the unwinding of helix II
is sufficient to disassemble the U4-U6 snRNA duplex
(5). If this is so, one possible function of helix II is to
hold indirectly the U4-U6 snRNA structure in place to antagonize
the premature formation of structures that are important for the
splicing reactions (i.e., U2-U6 snRNA helix Ia and the 3'-end
stem-loop of U6 snRNA). The unwinding of U2-U6 snRNA helix II
by a possible RNA helicase, such as Slt22p (53), may be a
crucial regulatory step in the initiation of splicing.
Interactions between the U2 and U5 snRNPs.
The genes
encoding the Slt factors were isolated on the basis of genetic
interaction with U2 snRNA, yet four of them are related functionally to U5 snRNA. This finding suggests that the U2
snRNA portion of U2-U6 snRNA helix II (the 5'-end
region), following resolution of this helix by Slt22p, may interact
with U5 snRNA. Three Slt factors are related to the tethering
function of U5 snRNA loop 1. Slt21p/Prp8p, which has been shown to
be cross-linked to both the 5' and 3' splice sites in human
(52) and yeast (45, 47, 48), may act to stabilize
the exon-U5 snRNA loop 1 interaction (45). This factor
is also involved in the recognition of the polypyrimidine tract
(46, 47) and 3'-splice-site selection (46). The
other two factors, Prp17p and Slu7p, are linked genetically to the
function of U5 snRNA loop 1 (14) and are in close
contact with the 3' splice site (1, 6, 13, 20, 48).
Further observations support the idea of interaction between the U2 and
U5 snRNPs. The failure of mutant slt22-1p to function, possibly because
of an inability to unwind U2-U6 snRNA helix II, results in the
accumulation of an unusual, "dead-end" splicing complex that lacks
the U5 snRNP (53). This suggests that the U2 snRNA part
of helix II may be involved in holding the U5 snRNP to the
spliceosome. A related experiment with a HeLa cell nuclear extract
demonstrated that an oligoribonucleotide complementary to U5 snRNA
(a region in the 3' side of loop 1) can induce the formation of a
U1-U4-U5 snRNP complex (3). This unusual U1-U4-U5 snRNP
complex may represent a transient step in spliceosome assembly during
which the U1-5'-splice-site and U4-U6 snRNA interactions are
disrupted and displaced by U6-5'-splice-site and U2-U6 snRNA interactions, respectively. In certain circumstances the U5 snRNP may
associate preferentially with the U1 and U4 snRNPs, which are in the
process of dissociating from the spliceosome (3). It is
known that residues in loop 1 of U5 snRNA can be cross-linked to
nucleotides of both pre-mRNA exons before and after the first step of
splicing (33, 44, 50, 52). We suggest that U5 snRNA may
become associated tightly with the spliceosome concomitantly with the
unwinding of U2-U6 snRNA helix II and that the 5' end of U2
snRNA may provide an RNA interaction(s) to anchor the U5 snRNA and/or to assist the U5 snRNA in the alignment of the two exons.
It has been proposed that the stem-loop structure of the U5 snRNA
loop 1 region is analogous to subdomain ID3 of autocatalytic group II
introns, which is essential for 5'-splice-site recognition and
tethering of the free 5' exon (32). However, unlike the loop
region of group II ID3, which contains the exon-binding site (EBS1)
complementary to the 3' end of the 5' exon (19),
uridine-rich U5 snRNA loop 1 may interact with the two exons by
noncanonical base pairing, since the exon sequences at both splice
sites are not conserved in pre-mRNAs. An additional RNA interaction(s)
may be necessary to stabilize the U5-exon interaction. In group II introns, a bulged region, ', at the bottom of the subdomain ID3 stem-loop, interacts with a region in subdomain IB, (15). Recently, an anchoring function for the tertiary
-' interaction has been suggested (16). Our data
(reference 53 and this study) may suggest a similar
role for the 5' end of U2 snRNA in binding the U5 snRNP to the
spliceosome, possibly in association with protein factors identified in
our genetic screen.
The observed genetic interactions between mutations in U2 and U5
snRNAs (Fig. 4) suggest a role for the helix Ib region of U2
snRNA in the second step: (i) this region of U2 snRNA may
interact with U5 snRNA to assist the tethering function of loop 1 and/or (ii) the 5' end of U2 snRNA (particularly nt 21, 22, and
23), in addition to that of U5 snRNA loop 1, may interact with
either or both exons to provide tethering. Since the U23 position of U2
snRNA is in close contact with the exon region of the 3' splice site (33), the second possibility is more likely and is
supported by the synthetic lethality of mutations in two second-step
factors (Prp17p and Slu7) with mutations in the 5' end of U2 snRNA
(Table 2).
Recently, Chiara et al. (9) have shown in the HeLa system
that cross-linking of the U2 snRNP protein, U2AF65, to the
polypyrimidine tract is replaced by cross-linking of three U5 snRNP
proteins, p110, p116, and p220 (the latter is an Slt21p/Prp8p ortholog)
prior to the second splicing step. This observation provides evidence
that an interaction (direct or indirect) between the U2 snRNP, bound to
the branchpoint site, and the U5 snRNP is important for positioning the
latter on the 3' splice site and thus for selection of the 3' splice
site. Our genetic data led to a similar conclusion.
Interactions among Slt factors: coordination of the two steps of
splicing.
Four slt mutations, slt11-1,
slt15/prp17-100, slt17/slu7-100, and
slt21/prp8-21, show pairwise synthetic lethality with each other and with prp16-1 (Fig. 4). prp16-1 was
identified initially as a suppressor of a mutation at the branchpoint
site; this mutation resulted in a second-step block due to the use of a
cryptic branchpoint site (7). McPheeters (29) has
shown genetic suppression of nonadenosine branchpoints by mutations in
two regions of U6 snRNA that are either part of U2-U6 snRNA
helix Ia (U57) or of the intramolecular stem-loop adjacent to helix Ib
(nt 63 to 65, 71, and 82 to 84) and that an allele of prp16,
prp16-302, is synthetically lethal with one of these U6
snRNA suppressors, U57C (29). Our results demonstrate
that prp16-1 is synthetically lethal with U2 substitutions in the U2-U6 snRNA helix Ia region (G26A).
The RNA-dependent ATPase activity of Prp16p is involved in remodeling
the spliceosome in order that the second-step reaction occurs
(43) and, analogously to what occurs with group
II self-splicing (18), that the lariat
intermediate is proofread (8). This suggestion of
proofreading was supported by the observation that replacement of
the adenosine residue at the branchpoint can block the second-step
reaction (37). These observations have led to the
suggestion that kinetic coordination of the two steps of pre-mRNA splicing is achieved by a mechanism in which the second step
serves as a trap for intermediates that have undergone productive
branching, thus driving the full splicing reaction to completion
(10).
Four Slt factors (Slt11p, Slt15p/Prp17p, Slt17p/Slu7p, and
Slt21p/Prp8p) as well as Prp16p may act as two units with
overlapping functions (Fig. 4). Through these potential
interactions, four components or functions of the spliceosome
that are important for the two steps of splicing are connected:
U2 snRNA (interacting with U6 snRNA and juxtaposing the 5'
splice site and branchpoint site), U5 snRNA (tethering the two
exons), Prp16p (proofreading the first-step reaction and
remodelling the spliceosome), and Slt17p/Slu7p plus
Slt21p/Prp8p (selection of the 3' splice site). Slt11p, required for
the first step of splicing, also interacts genetically with three
second-step factors (Fig. 4). These interactions may reflect a
mechanism that couples and coordinates the two steps of splicing.
We thank B. Funnell, S. Nouraini, and V. Lay for reading the
manuscript; J. D. Beggs, A. J. Newman, A. M. MacMillan,
and D. Jansma for comments and constructive discussion; R. W. van Nues, J. D. Beggs, T.-H. Chang, P. Raghunathan, C. Guthrie, D.-H. Kim, and R.-J. Lin for providing constructs and/or yeast strains; and U. Vijayraghavan for verifying the PRP17 sequence.
This research was supported by the National Cancer Institute of Canada
and the Medical Research Council of Canada.
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Abstract
Introduction
