Six novel genes necessary for pre-mRNA splicing in
Saccharomyces cerevisiae
Six novel genes necessary for pre-mRNA splicing in Saccharomyces cerevisiae
Janine R.
Maddock
+
,
Jagoree
Roy
[sect]
and
John L.
Woolford, Jr*
Department of Biological Sciences, Carnegie Mellon University,
Pittsburgh
, PA 15213,
USA
Received December 14, 1995;
Revised and Accepted February 7, 1996
ABSTRACT
We have identified six new genes whose products are necessary for the splicing
of nuclear pre-mRNA in the yeast
Saccharomyces cerevisiae
. A collection of 426 temperature-sensitive yeast strains was generated by EMS mutagenesis. These mutants
were screened for pre-mRNA splicing defects by an RNA gel blot assay, using the intron-containing
CRY1
and
ACT1
genes as hybridization probes. We identified 20 temperature-sensitive mutants defective in pre-mRNA splicing. Twelve appear to be allelic to the previously
identified
prp2, prp3, prp6, prp16/prp23, prp18, prp19
or
prp26
mutations that cause defects in spliceosome assembly or the first or second
step of splicing. One is allelic to
SNR14
encoding U4 snRNA. Six new complementation groups,
prp29
-
prp34
, were identified. Each of these mutants accumulates unspliced pre-mRNA at 37
o
C and thus is blocked in spliceosome assembly or early steps of pre-mRNA splicing before the first cleavage and ligation reaction. The
prp29
mutation is suppressed by multicopy
PRP2
and displays incomplete patterns of complementation with
prp2
alleles, suggesting that the
PRP29
gene product may interact with that of
PRP2
. There are now at least 42 different gene products, including the five
spliceosomal snRNAs and 37 different proteins that are necessary for pre-mRNA splicing in
Saccharomyces cerevisiae
. However, the number of yeast genes identifiable by this approach has not yet
been exhausted.
INTRODUCTION
Pre-mRNA splicing proceeds by assembly of splicing factors on pre-mRNA molecules to form spliceosomes, followed by intron excision and
exon joining. These complicated processes require numerous
trans
-acting protein factors and snRNAs (reviewed in
1
-
4
). The
cis
-acting sequences within pre-mRNAs required for splicing have been studied in detail (reviewed in
3
and
5
),
as have the roles of the U1, U2, U4, U5 and U6 snRNAs (
3
,
6
). Protein factors necessary for splicing have been identified by their capacity
to bind to pre-mRNA or by fractionation and reconstitution of active extracts from
mammalian cells (reviewed in
3
). snRNP particles have been purified and their protein constituents identified
(
7
). Mammalian spliceosomes purified by affinity chromatography using biotinylated
pre-mRNA contain at least 30 distinct proteins, including some proteins that
had previously been identified (
8
).
The power of genetic techniques in
Saccharomyces cerevisiae
provides a tool orthogonal to biochemical approaches to identify splicing
factors and to study their functions. Since spliceosome assembly and the
trans
-esterification reactions of splicing occur by similar steps in yeast and
metazoans, results obtained from the study of yeast splicing factors are of
general relevance. The first identification of yeast protein splicing factors
emerged from analysis of temperature-sensitive mutants defective in RNA synthesis (
9
,
10
). It was subsequently established that the primary defect in these
rna
mutants is lack of splicing of nuclear pre-mRNAs (
11
,
12
, reviewed in
13
). Thus these genes were renamed
prp
(
p
re-m
R
NA
p
rocessing) mutants,
prp2
-
prp10/11
(
prp10
and
prp11
are identical). Cloning of the
PRP2
-
PRP11
genes and subsequent characterization of
PRP
gene products and
prp
mutant extracts revealed that these Prp proteins are directly involved in
splicing (reviewed in
1
).
Additional yeast splicing factors have been discovered by searching for
pseudorevertants or multicopy suppressors of mutations in either
PRP
genes or introns, or for mutations synthetically lethal with mutations in genes
encoding small nuclear RNAs, or by identification of sequences in the genome
homologous to metazoan splicing factors (reviewed in
1
,
2
,
4
). Identification of mammalian homologues for several of these yeast proteins
confirms that the structure or function of these splicing factors is conserved
across species (
14
-
19
).
Analysis of the
prp2
-
prp11
mutants led to the identification of
bona fide
splicing factors. However, the search for
prp
mutants clearly was not saturated. Therefore, several laboratories initiated
additional screens of banks of temperature- or cold-sensitive mutants for those defective in pre-mRNA splicing. The
prp17-28, prp38-39
and
brr1-brr5
mutants were identified in
S. cerevisiae
and
prp1-3
in
S.pombe
(
20
-
24
; S. Noble and C. Guthrie, personal communication). We have carried out a
similar screen and identified temperature-sensitive mutants defective in pre-mRNA splicing that define six new genes,
PRP29
-
PRP34
.
MATERIALS AND METHODS
Strains, media and plasmids
JWY yeast strains were derived from strain A364A or from strains congenic to
A364A. These and other yeast strains used in this study are described in Table
1
. Yeast strains were grown in YEPD or defined synthetic medium lacking
appropriate supplements as described in (
25
). Diploids were sporulated on 1.5% agar medium containing either 0.1% dextrose,
0.25% yeast extract and 1.5% potassium acetate or 0.8% nutrient broth, 1% yeast
extract and 1% potassium acetate. Plasmids containing
PRP2
,
PRP3
,
SPP2
or
CRY1
were previously described (
26
-
28
). Plasmid pYACT1 (
29
) was kindly provided by John Abelson.
Genetic procedures
Methods for yeast mating, sporulation and tetrad analysis are described in (
30
). The temperature-sensitive mutants were scored by lack of growth on YEPD medium at 32 or 37oC. To determine complementation patterns for
prp
mutants, diploids produced by pair-wise mating with other putative splicing mutants or with
prp2-prp11,
prp17-prp27
and
prp38-prp39
strains were tested for growth on YEPD at 37oC. Complementation with
prp38
and
prp39
(
20
-
21
) was tested by Brian Rymond (personal communication). In some cases,
noncomplementing diploids were sporulated and tetrad progeny examined for segregation of temperature sensitivity.
Complementation between splicing-defective mutants that were not temperature sensitive for splicing defects was tested by
RNA blotting. Yeast cells were transformed with DNA by the spheroplast method (
31
) or by the LiAc protocol of ref.
32
.
Isolation of temperature-sensitive mutants
Wild-type yeast strain JWY70 was mutagenized with ethylmethane sulfonate (EMS),
as described in (
33
). Single colonies were patched onto YEPD plates and replica-plated to YEPD, and the plates incubated at 13, 23, 30, 32 or 37oC. Patches that grew slowly or failed to grow at 13 or 37oC were retained and frozen in 15% glycerol to create collections
of cold-sensitive and heat-sensitive mutants, respectively.
Isolation of RNA
RNA was isolated from yeast by a modification of the method of ref.
34
. A 3 ml culture of cells was grown in YEPD at 23oC to early log phase (1-2 * 10
7
cells/ml). For temperature shift experiments, half of each culture was shifted
to 37oC for 1-2 h, prior to extraction of RNA. Cells were pelleted by
centrifugation, suspended in 0.2 ml Kirby salts (
35
), and lysed by addition of 0.2 ml Kirby phenol plus one half volume glass beads
and subsequent vortexing. Organic and aqueous phases were separated by
centrifugation and the aqueous layer removed to a new Eppendorf tube. The
organic phase was extracted again with Kirby salts, and the two aqueous phases
pooled and extracted once more with phenol. The RNA was precipitated by ethanol
overnight at -20oC, collected by centrifugation in an Eppendorf microcentrifuge,
washed in 70% ethanol, dried
in vacuo
, and suspended in 30 [mu]l H
2
O.
Gel electrophoresis, blotting and hybridization of RNAs
Total RNA (~5 [mu]g) from each sample was subjected to gel electrophoresis and RNA blot
analysis as described in ref.
26
. Two intron-containing yeast genes,
CRY1
and
ACT1
, were used as hybridization probes. The 2.2 kb
Hin
dIII genomic DNA fragment containing
CRY1
(
26
) and the 2.2 kb
Eco
RI
-Hin
dIII genomic DNA fragment containing
ACT1
(
29
) were purified from plasmids pCRY1 and pYACT1, respectively, by restriction
enzyme digestion, gel electrophoresis and extraction with Gene Clean (Bio 101).
These DNA probes were radioactively labeled with
32
P by the random oligonucleotide primer method (
36
).
RESULTS
Isolation of temperature-sensitive lethal mutants
Identification of temperature sensitive mutants defective for pre-mRNA splicing
We screened the temperature-sensitive mutants for defects in pre-mRNA processing by an RNA blot hybridization assay using the
CRY1
or
ACT1
genes as probes.
CRY1
and
ACT1
each contain a single intron and encode abundant transcripts that are
efficiently spliced in wild-type cells (
26
,
37
). Unspliced
CRY1
or
ACT1
pre-mRNAs are readily detected by Northern blot assays of RNA extracted from
the
prp2-prp11
mutants grown at 23oC and shifted to 37oC for 1-2 h (
5
,
12
,
26
). To expedite screening of the 426 hs mutants, we first assayed only
CRY1
RNA from cultures grown at 23oC to mid-log phase and from cells shifted to 37oC for 1-2 h, using a rapid protocol for extracting RNA from small
volumes of culture. RNA blot data from a representative subset of the 426
mutants screened are shown in Figure
1
. Clearly, unspliced pre-mRNA or lariat intermediate accumulated and mRNA was diminished in many of
the mutants shifted to 37oC. In some cases, defects in splicing were less clear, due to underloading
or loading differences between 23 and 37oC samples, e.g. hs 68 or hs283. Other gels of these mutants clearly
demonstrate a splicing defect (data not shown). Those mutants in which
CRY1
mRNA splicing was defective at 37oC (Fig.
1
) were rescreened, using both
ACT1
and
CRY1
DNAs to probe RNA from cells grown at 23oC and from cells shifted to 37oC (Fig.
2
). Probes containing two different mosaic genes were used to increase the
likelihood of detecting mutants defective in the splicing machinery rather than
mutants in which the splicing of one transcript is specifically blocked (
38
,
39
). Mutants exhibiting no splicing phenotypes in either screen were discarded,
e.g. hs185, hs267, hs309, hs341, hs346, hs409 and hs425.
Genetic analysis
Each of the Ts
-
mutants defective in pre-mRNA splicing was mated to a wild type strain DBY1034 to create diploids.
Each of these diploids heterozygous for hs mutations grew well at 37oC and was wild type for pre-mRNA splicing, indicating that each mutation is recessive. The
resulting diploids were sporulated, and tetrads analyzed. Temperature
sensitivity and the splicing defects each segregated 2
+
:2
-
and co-segregated, indicating that the Ts
-
and splicing defects are, in each case, due to a mutation in a single nuclear
gene. Results for crosses of strains hs79, hs302 and hs337 to wild-type are shown in Figure
4
.
DISCUSSION
Screening collections of conditional lethal yeast mutants for those defective in
pre-mRNA splicing continues to yield novel genes. It is straightforward and
efficient: 3-5% of the Ts
-
mutants that have been screened in five independent collections are defective
in splicing (
10
,
20
,
22
,
24
, this work). Most (but not all) yeast splicing factors thus far identified are
essential, as one might expect. Thus screens of conditional lethal mutants
could yield many but not all potential yeast splicing factor genes. Some genes
may not be mutable to temperature or cold-sensitivity (
45
,
46
), or might be so small that the probability of identifying mutant alleles is
low. However, we did recover in our screen a mutant allele of
SNR14
encoding the 160 nucleotide long U4 snRNA.
Among the mutants we identified, 12 are apparently allelic to previously
identified
prp
mutants, one is allelic to
SNR14
encoding U4 snRNA, and seven are novel (Table
4
). As observed in previous screens, more alleles of
prp2
were found than for any other gene (
10
,
24
; Table
4
). The continued isolation of additional complementation groups and of only one
or two isolates of each suggests that these screens are not exhausted for Ts
-
splicing-defective mutants.
Some mutants may be only indirectly affected in splicing; e.g. the expression,
post-translational modification, or intracellular localization of splicing
factors may be deficient, rather than their function. Analysis of splicing
extracts derived from the Ts
-
mutants identifies those gene products directly involved in splicing. Like most
of the previously identified Ts
-
prp
mutants, extracts prepared from
prp31
or
prp33
mutants can be specifically heat-inactivated
in vitro
,
indicating that Prp31p and Prp33p are likely to be directly involved in splicing
(
50
; J. Roy, unpublished). Cloning and sequencing of
PRP31
and
PRP33
has verified that they are novel splicing factors (
50
; V. Lay, J. Roy, J. Woolford and J. Friesen, manuscript in preparation).
hs mutants that correspond to previously identified
prp
mutants
prp2
hs88
prp2-13
hs218
prp2-14
hs247
prp2-15
hs311
prp2-16
prp3
hs153
prp3-13
hs420
prp3-14
prp6
hs3
prp6-8
prp16
hs79
prp16-3
prp18
hs51
prp18-4
hs302
prp18-5
prp19
hs400
prp19-7
prp26
hs358
prp26-2
The majority of mutants we identified, including all those defining novel genes,
accumulate unspliced pre-mRNA at the nonpermissive temperature. These mutants may be blocked either
in different steps of spliceosome assembly or in splicing functions prior to
the first cleavage and ligation reaction. Mutants blocked in both early and
late steps of splicing might also accumulate pre-mRNA. Two mutants we identified, hs51 and hs302, are specifically blocked
in the second step of splicing and contain alleles of
prp18
. The hs358 mutant is blocked in intron turnover and may contain a mutant allele
of
prp26
. One potential class of splicing mutants not unambiguously distinguishable by
our RNA blot assay are those that fail to produce spliced mRNA, yet do not
accumulate unspliced pre-mRNA or splicing intermediates. Such might occur if splicing were blocked
at any early step prior to assembly of pre-mRNA with any splicing factors, such that unspliced pre-mRNA were degraded. Alternatively, pre-mRNA or splicing intermediates might be degraded in mutants in
which splicing complexes form but are disassembled.
Two results suggest that
prp29
(or its product) may interact with
PRP2
: (i) diploids formed between the
prp29
mutant (hs140) and some
prp2
strains grow poorly at 37oC. Noncomplementation of nonallelic genes is in some cases correlated with
functional or physical interactions between the gene products (
47
-
48
). (ii) Extra copies of
PRP2
suppress the Ts
-
phenotype of
prp29
. The
spp2
-
mutation is also suppressed by extra copies of
PRP2
(
27
). We have recently shown that Spp2p interacts with Prp2p and is necessary for
association of Prp2p with the spliceosome (
42
). Identification of other molecules that interact with Prp2p is of interest;
Prp2p is an RNA-dependent ATPase thought to be responsible for causing important changes
in the active site of the spliceosome prior to the first catalytic reaction of
splicing (
49
).
A wide array of splicing factors has been identified by genetic screens in
yeast, including snRNP proteins stably associated with the snRNAs and
spliceosomes, and proteins only transiently or weakly associated with
spliceosomes or snRNPs (reviewed in
1
). Many of these proteins might have been difficult to identify by conventional
biochemical approaches but can now be analyzed using the mutant strains.
Likewise, a different set of splicing factors has been identified by
fractionation of mammalian splicing extracts. Just as the similarities of the
basic mechanisms of pre-mRNA splicing in yeast and metazoans are becoming more apparent, so too
are the two sets of splicing proteins beginning to converge as more homologues
are identified. Nevertheless, new factors continue to be discovered.
ACKNOWLEDGEMENTS
We thank John Abelson, Christine Guthrie, Art Lustig, Evelyn Strauss and Usha
Vijayraghavan for
prp
strains, and John Abelson for plasmid pYACT1. We are grateful to Elaine
Weidenhammer and Elizabeth Jones for critical reading of the manuscript. This
work was supported by Public Health Service grant GM-38782 from the National Institutes of Health. J. L. Woolford, Jr was
supported by Research Career Development Award CA-01000 from the National Cancer Institute, and J. Roy was supported by
graduate fellowship award RCD-9154610 from the National Science Foundation.
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*
To whom correspondence should be addressed
Present addresses:
+
Department of Biology, University of Michigan, Ann Arbor, MI 48109, USA and
[sect]
Department of Microbiology and Immunology, University of California, San
Francisco, CA 94143-0414, USA
