Six Novel Genes Necessary for Pre-mRNA Splicing in Saccharomyces Cerevisiae (original) (raw)

Journal Article

,

Department of Biological Sciences, Carnegie Mellon University

,

Pittsburgh, PA 15213, USA

Search for other works by this author on:

,

Department of Biological Sciences, Carnegie Mellon University

,

Pittsburgh, PA 15213, USA

Search for other works by this author on:

Department of Biological Sciences, Carnegie Mellon University

,

Pittsburgh, PA 15213, USA

*To whom correspondence should be addressed

Search for other works by this author on:

+

Present address: Department of Biology, University of Michigan, Ann Arbor, MI 48109, USA

§

Present address: Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0414, USA

Author Notes

Received:

14 December 1995

Accepted:

07 February 1996

• PDF
• Split View
• • Article contents
  • Figures & tables
  • Video
  • Audio
  • Supplementary Data
• Cite

Cite

Janine R. Maddock, Jagoree Roy, John L. Woolford, Six Novel Genes Necessary for Pre-mRNA Splicing in Saccharomyces Cerevisiae, Nucleic Acids Research, Volume 24, Issue 6, 1 March 1996, Pages 1037–1044, https://doi.org/10.1093/nar/24.6.1037
Close

Navbar Search Filter Mobile Enter search term Search

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 introncontaining CRY1 and ACT1 genes as hybridization probes. We identified 20 temperaturesensitive 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°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 3,5), 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 (pre-mRNA processing) 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.

Table 1

Yeast strains used in this study

Materials and Methods

Strains, media and plasmids

JWY yeast strains were derived from strain A364A or from strains congenic to A364A. These and other yeas 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 37°C. 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 37°C. 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 37°C. Patches that grew slowly or failed to grow at 13 or 37°C were retained and frozen in 15% glycerol to create collections of cold-sensitive and heat-sensitive mutants, respectively.

Figure 1

Identification of splicing mutants by RNA blot hybridization. Mutants were grown to mid-logarithmic phase at 23°C; half of each culture was maintained at 23°C, and half shifted to 37°C for 1–2 h. RNA was extracted from each culture, resolved by agarose gel electrophoresis, blotted to Nytran and hybridized with the intron-containing CRY1 DNA labeled with 32P. Each hs mutant strain is indicated by a bracket above two lanes. Mutants containing alleles of previously identified PRP genes or novel PRP mutants are designated with PRP. A mutant containing an allele of SNR14 encoding U4 snRNA is also indicated. Those mutants not labeled with PRP are not defective in splicing or were subsequently discarded for other reasons. For each pair of lanes, RNA from cells grown at 23°C is on the left and that from cells shifted to 37°C is on the right. The positions of unspliced CRY1 pre-mRNA and spliced CRY1 mRNA are indicated. The differences in absolute amounts of RNA among samples most likely reflect differential yield of RNA, assayed by measuring ethidium bromide stained rRNA.

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 23°C to early log phase (1–2 × 107 cells/ml). For temperature shift experiments, half of each culture was shifted to 37°C 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 −20°C, collected by centrifugation in an Eppendorf microcentrifuge, washed in 70% ethanol, dried in vacuo, and suspended in 30 µl H2O.

Gel electrophoresis, blotting and hybridization of RNAs

Total RNA (∼5 mg) 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 32P by the random oligonucleotide primer method (36).

Results

Isolation of temperature-sensitive lethal mutants

Fourteen pools of wild-type yeast strain JWY70 were mutagenized with EMS under conditions that resulted in 50–80% killing. Approximately 20 000 independent colonies were picked and screened for acquisition of heat sensitivity (slow growth or no growth at 32 or 37°C) or cold sensitivity (slow or no growth at 13°C). The wild-type parental strain grew at all of these temperatures. 426 heat-sensitive (Ts−) mutants (hs1–hs426) and 194 cold-sensitive (Cs−) mutants (cs1–cs194) were identified among the different batches of mutagenized cells. Because the mutagenized populations of cells were allowed to recover for 2 h after mutagenesis, any two mutants within one pool could contain identical mutations.

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 23°C and shifted to 37°C 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 23°C to mid-log phase and from cells shifted to 37°C 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 37°C. In some cases, defects in splicing were less clear, due to underloading or loading differences between 23 and 37°C 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 37°C (Fig. 1) were rescreened, using both ACT1 and CRY1 DNAs to probe RNA from cells grown at 23°C and from cells shifted to 37°C (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.

Figure 2

Assaying splicing defects using both CRY1 and ACT1 probes. Cells were grown and RNA prepared and assayed as described in Figure 1. Mutant strains and CRY1 and ACT1 pre-mRNA and mRNA are indicated.

Of the 426 hs mutants screened, three phenotypic classes were observed in which splicing of both CRY1 and ACT1 mRNA was defective compared to wild-type cells. Most of these mutants accumulated CRY1 or ACT1 RNA that co-migrated on agarose gels with unspliced CRY1 or ACT1 pre-mRNA. Two mutants (hs51 and hs302) accumulated ACT1 or CRY1 RNA with electrophoretic mobilities intermediate to those of pre-mRNA and mRNA (Fig. 1). Primer extension analysis using oligonucleotides complementary to the 3′ exon and to the 5′ end of the intron of ACT1 and CRY1 demonstrated that these RNAs were intron-lariat 3′ exon splicing intermediates (data not shown). Mutant hs358 contained wild-type amounts of mRNA and no pre-mRNA after 1–2 h at 37°C, but accumulated a faster-migrating RNA species that was shown by primer extension using oligonucleotides complementary to the CRY1 and ACT1 introns to be the spliced intron-lariat product (Fig. 2 and data not shown).

The ratio of unspliced pre-mRNA to spliced mRNA varied considerably in mutants shifted to 37°C for 2 h. Although many of the mutants that accumulated pre-mRNA at 37°C contained diminished amounts of mRNA compared to cells grown at 23°C, several mutants contained near wild-type levels of spliced mRNA, e.g. hs35, hs197, hs209 and hs269 (Fig. 1). These may be particularly leaky mutants that are only partially defective in mRNA splicing. Upon testing some of these mutants, we observed that the splicing defect was exaggerated upon prolonged incubation at the nonpermissive temperature. One example (hs337) is shown in Figure 3. For this mutant, we also observed accumulation of intron lariat at later times after shift, suggesting a second defect in intron turnover or spliceosome disassembly. Several of the temperature-sensitive mutants that grew well at 23°C nevertheless exhibited modest defects in splicing at 23°C as well as a more severe splicing defect at 37°C. This phenotype is shown in Figure 1 for mutants hs51, hs140, hs190, hs194, hs236, hs247, hs302 and hs311.

Figure 3

Time course of accumulation of unspliced CRY1 and ACT1 pre-mRNA and diminution of spliced CRY1 and ACT1 mRNA in the hs337 prp32) mutant. The hs337 prp32) strain was grown to mid-log phase at 23°C and shifted to 37°C. At the indicated times, aliquots of cells were removed from the culture, and RNA was extracted, subjected to electrophoresis, blotted to Nytran and hybridized with 32P-labeled CRY1 and ACT1 DNAs. The positions of the CRY1 and ACT1 unspliced pre-mRNAs, spliced mRNAs and intron-lariat products are indicated.

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 37°C 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.

Figure 4

Co-segregation of pre-mRNA splicing defects and temperature sensitivity of the hs mutants in a cross to wild-type yeast. Ts− mutants hs302, (prp18), hs337 (prp32) and hs79 (prp16) were mated to wild-type strain DBY1034, diploids were sporulated, and tetrads dissected. RNA was extracted from cultures of each spore clone grown to mid-log phase at 23°C and shifted to 35°C for 2 h, and assayed for splicing defects by RNA blotting and hybridization with 32P-labeled CRY1 DNA. Unspliced CRY1 pre-mRNA and spliced CRY1 mRNA are indicated, as is the snRNA snR189 that was used as a loading control.

To determine whether the hs mutants contain mutations in novel PRP genes or are new alleles of previously identified PRP loci, complementation tests were performed (Tables 2 and 3). In most cases where noncomplementation was observed, the diploids were sporulated and tetrads were analyzed to determine whether the noncomplementing mutations were linked. The five mutants hs283, hs29, hs337, hs68 and hs89 complemented each other, all of the other hs mutants, and the previously identified prp mutants prp2–prp11, prp16–prp24, prp27 and prp38–prp39. hs87 and hs367 complemented all other mutants, but failed to complement each other. Crosses indicate hs87 and hs367 contain tightly linked or identical mutations conferring a Ts− phenotype. We conclude that each of the hs283, hs29, hs337, hs68 and hs87/hs367 strains contains a mutation in a single nuclear gene and defines one of five new genes necessary for pre-mRNA splicing, which we designate prp30–prp34, respectively. A Northern blot showing the splicing defect of each of these mutants shifted from 23 to 37°C for 2 h is shown in Figure 5.

Table 2

Complementation tests between hs mutants and prp mutants1

Table 3

Complementation tests between hs mutants defective in pre-mRNA splicing

The wild-type gene corresponding to the hs89 mutant allele was cloned by transformation and complementation of the Ts−phenotype. We confirmed that the cloned DNA originated from the same genomic locus as the Ts− mutation by integrating the cloned DNA at its homologous locus and analyzing linkage of the integrated URA3 marker to the Ts− phenotype (40, data not shown). The smallest complementing subclone derived from the original plasmid contains a 0.9 kb _Cla_I_-Eco_RV fragment bearing the SNR14 gene encoding U4 snRNA (41). Thus hs89 may contain a Ts− allele of U4 snRNA. Consistent with this finding, we identified the SNR6 gene encoding U6 snRNA as a multicopy suppressor of hs89 (40).

The temperature-sensitive phenotype of the hs88, hs218, hs247 and hs311 strains was complemented in crosses to all of the other hs mutants and to prp3–prp11, prp16–prp24, prp27 and prp38–prp39 mutants, but was not complemented in crosses to prp2 mutants (Tables 2 and 3). In addition, hs311 failed to complement hs88, hs218 or hs247 (Table 3). The temperature sensitivity of these four hs mutants was specifically complemented upon transformation with YCp50-borne PRP2 (28) and was efficiently suppressed by introduction of one or a few extra copies of SPP2, a gene dosage suppressor of prp2 (42). Analysis of crosses of each of these hs mutants to a prp2 strain indicated that each mutation conferring a Ts−phenotype was tightly linked to prp2. We conclude that hs88, hs218, hs247 and hs311 contain Ts− alleles of prp2.

Crosses of hs140 to all of the prp mutants except prp2 revealed efficient complementation of the Ts− phenotype at 37°C. Complementation between hs140 and prp2 was more complex; hs140/prp2 diploids did not grow as well at 37°C as wild-type controls or diploids formed between hs140 and other prp mutants. To determine whether the weak complementation between hs140 and prp2 was specific to certain alleles of prp2, seven other previously identified prp2 isolates (9) as well as hs88, hs218, hs247 and hs311 were mated to hs140. Some combinations of hs140/prp2 diploids grew well at 37°C while others did not (43; Tables 2 and 3). Analysis of the meiotic segregation of Ts− phenotypes of hs140 and several different prp2 isolates indicated that the mutation in hs140 conferring a Ts− phenotype is linked to prp2 but is in a different gene. Fourteen parental ditype, one nonparental ditype and six tetratype tetrads were recovered from 21 four-spored tetrads, indicating that the mutation in hs140 is 29 cM from prp2. The Ts− phenotype of hs140 was efficiently suppressed at 37°C upon transformation with Ycp50-borne PRP2. However, extra copies of SPP2, a multicopy suppressor of prp2 (27,42), did not suppress the Ts− phenotype of hs140. We conclude that hs140 contains a mutation in a novel gene, which we designate prp29, that fails to efficiently complement the Ts−phenotype of some alleles of prp2.

We observed complementation between mutants hs153 or hs420 and all of the other hs mutants and prp mutants except prp3 (Tables 2 and 3). The Ts− phenotype of hs153 was complemented by transformation of YCp50 containing PRP3 (28); hs420 was not tested. Thus hs153 contains a mutation most likely allelic to prp3, and hs420 may as well. hs3 was identified as a possible allele of prp6 by both complementation tests and molecular cloning. hs51 and hs302 specifically did not complement prp18, and hs400 did not complement prp19. hs79 failed to complement prp16 and prp23, which are identical to each other (44).

Both the intron-accumulation and the temperature-sensitive phenotypes of mutant hs358 segregated 2+:2− in crosses to wild-type, but did not cosegregate (data not shown). Thus a nonlethal mutation caused accumulation of spliced intron RNA in strain hs358 and was unlinked to the temperature-sensitive mutation in that strain. For this reason, complementation tests were done between hs358 and the intron-accumulating prp25 and prp26 mutants by assaying phenotypes of the diploids on RNA blots. Mutant hs358 failed to complement prp26 but did complement prp25. Linkage analysis will be necessary to distinguish whether hs358 is an allele of prp26 or exhibits unlinked noncomplementation with prp26.

Figure 5

Each of the temperature-sensitive mutants prp30-prp34 accumulates unspliced pre-mRNA when shifted to the nonpermissive temperature for 2 h. Strains hs283 prp30), hs29 prp31), hs337 prp32), hs68 prp33), hs87 prp34), as well as wild type and prp2 strains were grown to mid-log phase at 23°C. Half of each culture was shifted to 37°C for 2 h. RNA was extracted from each, subjected to electrophoresis, blotted and probed with 32P-labeled CRY1 DNA. CRY1 pre-mRNA and mRNA, as well as snR189 used as a loading control, are indicated.

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 aie 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).

Table 4

hs mutants that correspond to previously identified prp mutants

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 37°C. Noncomplementa-tion 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 PRRP2 (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.

References

1

Splicing factors in the yeast Saccharomyces cerevisiae

,

Eukaryotic mRNA Processing, Frontiers in Molecular Biology

,

1993

Oxford, UK

Oxford University Press

2

,

Bull. Inst. Pasteur

,

1994

, vol.

92

(pg.

153

-

179

)

3

Splicing of precursors to messenger RNA by the spliceosome

,

The RNA World

,

1993

Cold Spring Harbor, NY

Cold Spring Harbor Laboratory Press

(pg.

303

-

357

)

4

Yeast pre-mRNA splicing

,

The Molecular Biology of the Yeast Saccharomyces cerevisiae: Gene Expression

,

1992

Cold Spring Harbor, NY

Cold Spring Harbor Laboratory Press

(pg.

143

-

192

)

5

,

Yeast

,

1989

, vol.

5

(pg.

439

-

457

)

6

,

Annu. Rev. Genet.

,

1994

, vol.

28

(pg.

1

-

26

)

7

,

Biochim. Biophys. Acta

,

1990

, vol.

1087

(pg.

265

-

292

)

8

,

Genes Dev.

,

1992

, vol.

6

(pg.

1986

-

2000

)

9

,

J. Bacteriol.

,

1967

, vol.

93

(pg.

1662

-

1670

)

10

,

Mol. Gen. Genet.

,

1970

, vol.

109

(pg.

42

-

56

)

11

,

Cell

,

1986

, vol.

47

(pg.

953

-

963

)

12

,

Cell

,

1981

, vol.

24

(pg.

679

-

686

)

13

,

Genes Dev.

,

1987

, vol.

1

(pg.

1

-

3

)

14

,

Nature

,

1989

, vol.

34

(pg.

819

-

821

)

15

,

Science

,

1993

, vol.

262

(pg.

105

-

108

)

16

,

Science

,

1993

, vol.

262

(pg.

102

-

105

)

17

,

RNA

,

1995

, vol.

1

(pg.

260

-

272

)

18

,

Nucleic Acids Res.

,

1993

, vol.

21

(pg.

3501

-

3505

)

19

,

Nucleic Acids Res.

,

1994

, vol.

22

(pg.

4510

-

4519

)

20

,

Mol. Cell. Biol.

,

1992

, vol.

12

(pg.

3939

-

3947

)

21

,

Mol. Cell. Biol.

,

1994

, vol.

14

(pg.

3623

-

3633

)

22

,

EMBO J.

,

1989

, vol.

8

(pg.

551

-

559

)

23

,

Genes Dev.

,

1991

, vol.

5

(pg.

629

-

641

)

24

,

Genes Dev.

,

1989

, vol.

3

(pg.

1206

-

1216

)

25

,

Methods in Yeast Genetics

,

1986

Cold Spring Harbor, NY

Cold Spring Harbor Laboratory Press

26

,

Nucleic Acids Res.

,

1983

, vol.

11

(pg.

403

-

420

)

27

,

Genetics

,

1987

, vol.

117

(pg.

619

-

631

)

28

,

Mol. Cell. Biol.

,

1984

, vol.

4

(pg.

2396

-

2405

)

29

,

Proc. Natl. Acad. Sci. USA

,

1980

, vol.

77

(pg.

3912

-

3916

)

30

,

Yeast Genetics

,

1969

, vol.

1

NY

Academic Press Inc.

(pg.

385

-

460

)

The Yeasts

31

,

Nature

,

1978

, vol.

275

(pg.

104

-

109

)

32

,

J. Bacteriol

,

1983

, vol.

153

(pg.

163

-

168

)

33

,

Genetics

,

1988

, vol.

119

(pg.

13

-

20

)

34

,

Cell

,

1977

, vol.

10

(pg.

453

-

462

)

35

,

Biochem. J.

,

1965

, vol.

96

(pg.

266

-

269

)

36

,

Anal. Biochem.

,

1983

, vol.

13

(pg.

6

-

13

)

37

,

Cell

,

1984

, vol.

39

(pg.

611

-

621

)

38

,

Cell

,

1991

, vol.

66

(pg.

1257

-

1268

)

39

,

Mol. Cell. Biol.

,

1991

, vol.

11

(pg.

5693

-

5700

)

40

,

1995

Carnegie Mellon University

Ph.D. Thesis

41

,

Cell

,

1987

, vol.

50

(pg.

585

-

592

)

42

,

RNA

,

1995

, vol.

1

(pg.

375

-

390

)

43

,

1990

Carnegie Mellon University

Ph.D. Thesis

44

,

Nature

,

1991

, vol.

349

(pg.

494

-

499

)

45

,

Genetics

,

1991

, vol.

127

(pg.

279

-

285

)

46

,

Genetics

,

1984

, vol.

108

(pg.

67

-

90

)

47

Genetic analysis of microtubule function in Drosophila

,

Molecular Biology of the Cytoskeleton

,

1984

Cold Spring Harbor, NY

Cold Spring Harbor Laboratory Press

48

,

Genetics

,

1988

, vol.

119

(pg.

249

-

260

)

49

,

Nucleic Acids Res.

,

1990

, vol.

21

pg.

6447

50

,

Nucleic Acids Res.

,

1996

, vol.

24

(pg.

1164

-

1170

)

Author notes

Present address: Department of Biology, University of Michigan, Ann Arbor, MI 48109, USA

§

Present address: Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0414, USA

© 1996 Oxford University Press

I agree to the terms and conditions. You must accept the terms and conditions.

Submit a comment

Name

Affiliations

Comment title

Comment

You have entered an invalid code

Thank you for submitting a comment on this article. Your comment will be reviewed and published at the journal's discretion. Please check for further notifications by email.

Citations

Views

Altmetric

Metrics

Total Views 808

386 Pageviews

422 PDF Downloads

Since 12/1/2016

Citations

17 Web of Science

×

Email alerts

Citing articles via

• Latest

• Most Read

• Most Cited

More from Oxford Academic