The Yeast Splicing Factor Prp40p Contains Functional Leucine-Rich Nuclear Export Signals That Are Essential for Splicing

Corresponding author: Paul G. Siliciano, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church St., Minneapolis, MN 55455., pauls{at}lenti.med.umn.edu (E-mail)

Communicating editor: M. HAMPSEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

To investigate the function of the essential U1 snRNP protein Prp40p, we performed a synthetic lethal screen in Saccharomyces cerevisiae. Using an allele of PRP40 that deletes 47 internal residues and causes only a slight growth defect, we identified aphenotypic mutations in three distinct complementation groups that conferred synthetic lethality. The synthetic phenotypes caused by these mutations were suppressed by wild-type copies of CRM1 (XPO1), YNL187w, and SME1, respectively. The strains whose synthetic phenotypes were suppressed by CRM1 contained no mutations in the CRM1 coding sequence or promoter. This indicates that overexpression of CRM1 confers dosage suppression of the synthetic lethality. Interestingly, PRP40 and YNL187w encode proteins with putative leucine-rich nuclear export signal (NES) sequences that fit the consensus sequence recognized by Crm1p. One of Prp40p's two NESs lies within the internal deletion. We demonstrate here that the NES sequences of Prp40p are functional for nuclear export in a leptomycin B-sensitive manner. Furthermore, mutation of these NES sequences confers temperature-sensitive growth and a pre-mRNA splicing defect. Although we do not expect that yeast snRNPs undergo compartmentalized biogenesis like their metazoan counterparts, our results suggest that Prp40p and Ynl187wp contain redundant NESs that aid in an important, Crm1p-mediated nuclear export event.

NUCLEAR pre-mRNA splicing requires five small nuclear ribonucleoprotein particles (snRNPs) and a number of soluble protein factors (BURGE et al. 1999 ). Early during spliceosome assembly, the U1 snRNP binds to the 5' splice site to form a complex that is committed to the splicing pathway (the commitment complex). Subsequently, the U2 snRNP binds in an ATP-dependent manner to form the prespliceosome complex. The U4/U6-U5 tri-snRNP then joins this complex, which undergoes a series of conformational rearrangements to form an active spliceosome.

Each snRNP is composed of a small nuclear RNA (snRNA) that associates with a specific set of proteins (LUHRMANN et al. 1990 ; ZIEVE and SAUTERER 1990 ). Several of these proteins are common to the U1, U2, U4, and U5 snRNPs and are known collectively as the Sm proteins. The Sm proteins are involved in snRNP stability and binding of the U1 snRNP to the 5' splice site (ZHANG et al. 2001 ). In addition to the Sm proteins, each snRNP contains a set of unique proteins.

The metazoan U1 snRNP contains the U1-specific proteins U1-70K, U1-A, and U1-C. The Saccharomyces cerevisiae U1 snRNP includes the homologs of these proteins as well as seven additional proteins that have not been detected in the metazoan U1 snRNP (NEUBAUER et al. 1997 ; GOTTSCHALK et al. 1998 ; MCLEAN and RYMOND 1998 ; FORTES et al. 1999 ). One of these yeast-specific U1 snRNP proteins is Prp40p, which was first identified as a suppressor of U1 snRNA mutations (KAO and SILICIANO 1996 ). Biochemical analysis shows that Prp40p is a stable component of the U1 snRNP and is essential for splicing in vitro and in vivo (KAO and SILICIANO 1996 ). Prp40p interacts with the conserved splicing factors branchpoint bridging protein (BBP), Prp8p, and Mud2p through its amino-terminal WW domains (ABOVICH and ROSBASH 1997 ) as well as with Prp5p and other proteins required for prespliceosome formation (K. SAKAI and P. G. SILICIANO, unpublished data). Analysis of spliceosome assembly in vitro using prp40 mutant extracts revealed that the U1 snRNP can still bind the 5' splice site in the absence of active Prp40p. However, inactivation of Prp40p blocks the addition of the U2 snRNP to the commitment complex (K. SAKAI and P. G. SILICIANO, unpublished data). These experiments demonstrate that Prp40p participates directly in splicing at the step of prespliceosome formation.

In metazoans, snRNP biogenesis is a compartmentalized event requiring the snRNA to traverse the nuclear envelope twice (reviewed in KOMEILI and O'SHEA 2001 ; KUERSTEN et al. 2001 ; WILL and LUHRMANN 2001 ). During transcription, the snRNA receives a 7-methyl-guanosine cap. Immediately after synthesis, the snRNA is exported to the cytoplasm by the conserved export factor Crm1p (HAMM and MATTAJ 1990 ; FORNEROD et al. 1997A ). The accessory protein PHAX is also required and acts as an adapter between the 5' cap of the snRNA and Crm1p (OHNO et al. 2000 ). While in the cytoplasm, the nascent snRNA associates with the Sm proteins and its cap becomes hypermethylated (MATTAJ 1986 ). In addition, the 3' end of U1 snRNA is cleaved by exonucleases and some of the unique proteins associate with the maturing snRNP. The snRNP is then imported to the nucleus by Snurportin1, where it completes its maturation and participates in splicing (HUBER et al. 1998 ).

While compartmentalized snRNP biogenesis has been well characterized in metazoans, nuclear export of nascent snRNAs and compartmentalized snRNP biogenesis has not been demonstrated in S. cerevisiae. In fact, there is compelling evidence that compartmentalized biogenesis does not occur in yeast. First, no snRNAs can be detected in the yeast cytoplasm (data not shown and C. GUTHRIE, personal communication). Second, cap hypermethylation and 3' end trimming of snRNAs are nuclear restricted in yeast (VAN HOOF et al. 2000 ; MOUAIKEL et al. 2002 ). Third, there is no obvious homolog of the metazoan snRNP export and import factors, PHAX and Snurportin1, respectively (HUBER et al. 1998 ; OHNO et al. 2000 ). Last, all essential proteins of the yeast U1 snRNP appear to contain their own nuclear localization signals. Yeast do, however, contain an essential homolog of Crm1p that is required for other nuclear export events.

Crm1p, also known as Exportin-1 (Xpo1p) is a large, well-conserved protein that was first identified in Schizosaccharomyces pombe (ADACHI and YANAGIDA 1989 ). It mediates the nuclear export of protein cargoes through the nuclear pore complex in a saturable, signal- and energy-dependent manner (reviewed in KOMEILI and O'SHEA 2001 ; OSSAREH-NAZARI et al. 2001 ; WEIS 2002 ). Crm1p functions through interaction with leucine-rich nuclear export signal (NES) sequences in its cargoes (FORNEROD et al. 1997A ; STADE et al. 1997 ). Mutation of individual leucine residues in an NES-containing reporter protein abolishes export in yeast. Furthermore, a yeast strain containing the temperature-sensitive crm1-1 mutation loses its ability to export this reporter at the restrictive temperature (STADE et al. 1997 ). This requirement for the NES appears to represent a direct physical interaction between the cargo and Crm1p (FORNEROD et al. 1997A ; NEVILLE et al. 1997 ; OSSAREH-NAZARI et al. 1997 ; STADE et al. 1997 ). Furthermore, the cytotoxin, leptomycin B, specifically inhibits Crm1p-mediated nuclear export by covalent attachment to a cysteine residue in Crm1p (NISHI et al. 1994 ; WOLFF et al. 1997 ; KUDO et al. 1998 , KUDO et al. 1999 ).

In addition to transporting proteins, Crm1p has been shown to transport RNA cargoes across the nuclear envelope (FISCHER et al. 1995 ; STUTZ and ROSBASH 1998 ). Importantly, Crm1p does not bind its RNA cargoes directly. Instead, it binds an NES-containing adapter protein, which in turn binds the RNA. For example, Crm1p is required for the human immunodeficiency virus-1 Rev-mediated nuclear export of Rev response element (RRE)-containing RNAs in Xenopus oocytes (FORNEROD et al. 1997A ) and in S. cerevisiae (NEVILLE et al. 1997 ). In this instance, Rev acts as an NES-containing adapter protein that binds RRE-containing RNAs and promotes their export by Crm1p (NEVILLE et al. 1997 ; STUTZ and ROSBASH 1998 ).

Specificity for loading and unloading of Crm1p cargoes is controlled by the small GTP-binding protein Gsp1p (the yeast homologs of Ran). In the nucleus, the GTP-bound form of Gsp1p cooperatively forms a heterotrimeric complex with Crm1p and the NES-containing cargo protein. In the cytoplasm, Gsp1p hydrolyzes GTP to GDP and the complex dissociates, depositing the cargo in the cytosol. Crm1p is then recycled back to the nucleus via classical nuclear import pathways (reviewed in KOMEILI and O'SHEA 2001 ; OSSAREH-NAZARI et al. 2001 ; WEIS 2002 ).

Here, we used a synthetic lethal screen to identify genes that interact with PRP40 and discovered a link between this U1-specific splicing factor and the Crm1p-mediated export pathway. One class of synthetic lethal mutations was suppressed by CRM1 overexpression. A second class of synthetic lethal mutations was null alleles of a nonessential, NES-encoding gene. Importantly, Prp40p contains two leucine-rich regions that fit the NES consensus sequence (BOGERD et al. 1996 ). We demonstrate that these two NES sequences interact genetically with each other and the synthetic lethal mutations. Moreover, they are required for efficient pre-mRNA splicing. We further show that a Prp40p-GFP fusion protein, present in both the nucleus and the cytoplasm, becomes almost exclusively nuclear upon mutation of the NES sequences. Addition of leptomycin B mimics this nuclear accumulation, indicating that Crm1p function is required for this shuttling. These genetic and biochemical observations suggest a model in which Prp40p export from the nucleus is required for pre-mRNA splicing.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains and growth conditions:
All yeast strains in this study are isogenic with TR2 (PARKER et al. 1988 ) and are listed in Table 1. Yeast transformations were performed as previously described (SCHIESTL and GIETZ 1989 ). Media, mating, sporulation, and tetrad dissection were carried out according to standard methods that have also been previously described (GUTHRIE and FINK 1991 ).

Plasmid construction:
The internal deletions of region 1, region 2, ARM, and the K4-A4 mutation were all generated in PRP40 by transformer mutagenesis (CLONTECH, Palo Alto, CA) using oligonucleotides PRBD1 (5'-ccaacgggtgtaatatttgtcatcatcgaccatcc-3'), RBD2-40 (5'-gttgttataataccgttttcgtcggcaattagtc-3'), ARM (5'-gcttttaaagttatcgagctcatttagtttg-3'), and MM40-K4 (5'-ggg cct gtg ttt cca att ctt ctt ctg att ctt tct gag-3'), respectively. The mutations were confirmed by sequencing. The prp40 alleles for high-copy plasmids were generated by PCR using oligonucleotides creating a BamHI site immediately 5' to the start codon (PRP40) and a BamHI site and consensus start codon at D256 (prp40256) with oligonucleotides 40AUG2Fbam (5'-cgggatccctatgtctatttggaaggaagc-3')/643'U (5'-tgcggtgatcgcaaacttga-3') and NESpeptideAUG (5'-gcggatccaccatggtagataccctcatcgacactc-3')/643'U, respectively. Using the endogenous downstream SalI site, these products were digested with BamHI and SalI and cloned into the high-copy vector PG-1 (TRP1 2µ; GUTHRIE and FINK 1991 ) 3' of the GPD promoter. The gene encoding enhanced green fluorescent protein (EGFP) was PCR amplified from the mammalian expression vector pEGFP-C3 (CLONTECH), creating 5' and 3' NotI sites, using oligonucleotides GFPAUG (5'-gaggaggaaggcggccgcatggtgagcaagggcgagg-3') and GFPTCA (5'-gagaggagaagcggccgctcagttatctagatccggtgg-3'). Products were digested with NotI and cloned into a NotI site introduced at the penultimate codon of the PRP40 (KAO and SILICIANO 1996 ).

CRM1 was subcloned as a SalI fragment from a YPH1 genomic library (American Type Culture Collection no. 77164) into the vector pSE360 (URA3 CEN). The leptomycin B (LMB)-sensitive allele (crm1-lmb) was created using transformer mutagenesis (CLONTECH) to replace T539 with C539 (KUDO et al. 1999 ; NEVILLE and ROSBASH 1999 ). This mutation was confirmed by sequencing.

Synthetic lethal screen:
Strain 40SS containing pSE358-prp402 (TRP1 CEN) and pSE360-PRP40 (URA3 CEN) was grown to midlog phase, diluted to 10,000 cells per ml, and 150 µl of this dilution was spread onto minimal plates lacking tryptophan and uracil. Forty plates were exposed to ultraviolet (UV) light (15 mJ) in a Stratalinker 1800 (Stratagene, La Jolla, CA) in the dark. Immediately following mutagenesis, the plates were wrapped in aluminum foil and incubated at 30° for 48 hr. This exposure resulted in a 78% kill rate. A second screen was performed using 10 mJ, which resulted in a 55% kill rate.

After 2 days at 30°, the mutagenized plates were replica plated to plates containing 5-fluoroorotic acid (5-FOA; Diagnostic Chemicals, Oxford, CT). These plates were allowed to grow at 24° for 2 days and then replicated to a second 5-FOA plate. After 2 more days, candidate synthetic lethal strains were chosen on the basis of their apparent death on the second 5-FOA plate. Candidate strains were picked from the original mutagenesis plates by comparison to the second 5-FOA plate and retested to confirm 5-FOA sensitivity. These were named synthetic lethal with forty (slf).

To allow loss of the TRP1 plasmids, pre-5-FOA strains were grown in liquid YEPD media overnight at 30°, diluted, and plated on YEPD. After 2 days growth at 30°, the plates were replica plated to -trp and to YEPD plates. Trp- cells were chosen for a trans test. These strains were transformed with an unmutagenized pSE358-prp402 plasmid to verify that the synthetic lethality resulted from a genomic rather than a plasmid-borne mutation. These strains were also transformed with plasmids carrying PRP40, prp40ARM, or prp40K4-A4 to determine allele specificity. Four transformants of each strain were patched to a master -trp -ura plate and tested on 5-FOA plates as above.

To remove possible mutations in other genes, the synthetic lethal strains were backcrossed to 40SS. After the first cross, strains of both mating types for each of the slf mutants were recovered. pRS317-prp402 (LYS2 CEN) was transformed into the slf strains that had lost their TRP1 vectors. These strains were then mated to slf a strains containing pSE358-prp402. To test for dominance, diploids were selected, allowed to lose their TRP1 vectors, and tested for growth on 5-FOA plates.

Suppressors of the slf synthetic phenotypes were isolated from a YPH1 genomic library in pRS200 (TRP1 CEN; American Type Culture Collection no. 77164). The slf mutant strains were transformed with this library and plasmids that conferred 5-FOA resistance were isolated. Plasmids recovered from 5-FOA-resistant strains were sequenced and each open reading frame was subcloned and tested individually for 5-FOA resistance.

In vivo splicing assay:
Strain 40SS was transformed with alleles of prp40 in pSE358 and cured of pSE360-PRP40 by 5-FOA treatment. Cells were grown overnight in YEPD media to midlog phase at 30°. Cultures were then shifted to a 37° shaking water bath. Aliquots taken prior to and after temperature shift were frozen at -80° until total RNA was harvested by the hot phenol method previously described (DOMDEY et al. 1984 ). Primer extensions were performed as previously described (FRANK and GUTHRIE 1992 ), using 12 µg of total RNA and primer U3exon2 (5'-ccaagttggattcagt-3') or RPL30A (5'-gtggacttgtaacctaaggtg-3').

Fluorescence microscopy:
Cells were grown overnight at 30° in liquid minimal media to midlog phase prior to fixation. For leptomycin B experiments, the culture was split and leptomycin B in ethanol or ethanol alone was added to the cultures at a final concentration of 100 ng/ml leptomycin B, 1% ethanol or 1% ethanol, respectively, for 45 min. Leptomycin B was a kind gift from Minoru Yoshida (NISHI et al. 1994 ).

All cells were fixed in 3.7% formaldehyde for 1 hr and washed twice with PBS (40 mM K₂HPO₄, 10 mM KH₂PO₄, 150 mM NaCl pH 7.5) prior to mounting for 5 min on poly-L-lysine-coated slides. Slides were then washed three times in PBS, dried, immersed in ice-cold methanol for 6 min, immersed in ice-cold acetone for 30 sec, washed four times with PBS, and covered with mounting media containing 22.5 ng/ml 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and a coverslip. Fluorescent cells were visualized using a Zeiss Axioplan II imaging microscope with a 63x Plan-Apochromat lens. Images were acquired with a Diagnostics Instruments Spot CCD camera using Diagnostics Instruments Spot software. The average nuclear and cytoplasmic levels of fluorescence of a 25-pixel area (about 1/100th of the nucleus) were determined by using the eye-dropper tool in Adobe Photoshop software.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Prp40p contains two regions with homology to the double-stranded RNA-binding domain (dsRBD; ST. JOHNSTON et al. 1992 ). In other proteins, these motifs bind double-stranded RNA (dsRNA) in vitro in a sequence-independent manner (BASS 1995 ). However, all attempts to show RNA binding by Prp40p have proven unsuccessful (data not shown). Therefore, we call these sequences in Prp40p region 1 and region 2 (Fig 1A). To investigate the functions of these regions, we made in-frame deletions of each. Precise deletion of region 1 caused lethality (data not shown) while deletion of region 2 resulted in a slight growth defect at all temperatures (Fig 1B and data not shown). This slow-growth phenotype indicates that region 2 is important but not essential for some aspect of Prp40p function.

View larger version (29K): In this window In a new window Download PPT slide   Figure 1. Domains of Prp40p. (A) Domains of Prp40p are indicated with their positions in the amino acid sequence given. WW represents the WWP domain, ARM represents the arginine-rich motif. (B) The phenotypes of prp40 mutations are given. Region 2 K4-A4 changes four lysine residues to alanines. ARM deletion deletes the sequence 348-RLRNYTRDRIAR-359. (C) Comparison of Prp40p and Ynl187wp NES sequences to the NES consensus sequence (BOGERD et al. 1996 ). Mutant sequences show amino acid substitutions used in this study.

A synthetic lethal screen for genes that interact with PRP40:
To investigate the function of region 2, we sought to identify genes whose products interact with this region. To do so, a synthetic lethal screen was performed using the region 2 deletion (prp402) as a starting mutation. Synthetic lethal screens detect the synergistic defects that result when a cell contains mild mutations in two functionally related genes (FRANK et al. 1992 ; LIAO et al. 1993 ). Thus, a synthetic lethal interaction between two genes is predictive of important functional interactions between those genes.

We started with the strain 40SS, in which the chromosomal copy of PRP40 was disrupted and complemented by wild-type PRP40 carried on a URA3 plasmid. The strain also contained a second plasmid carrying TRP1 and prp402. This strain was mutagenized with UV light and the surviving cells were allowed to form colonies. These colonies were then replica plated to 5-FOA-containing media to select cells that had lost the PRP40 URA3 plasmid. These cells depended on prp402 as their sole source of Prp40p. If any of these cells contained a UV-induced genomic mutation that was synthetically lethal with prp402, they did not survive the 5-FOA treatment. We identified these candidates and recovered them from the pre-5-FOA replica plate.

To ensure that the UV mutations caused specific, synergistic defects with prp402, only UV-induced mutations that had no growth defect in the presence of wild-type PRP40 were pursued (Table 2). By studying only those UV-induced mutations that caused no detectable phenotype on their own, we avoided false positives caused by the additive phenotypes of unrelated mutations. For this test, strains from the pre-5-FOA plates were evaluated. These strains still had a wild-type copy of PRP40 and displayed only the defect caused by the UV-induced mutation. The rate of colony formation of these synthetic lethal strains was compared to that of a wild-type strain at five different temperatures: 18°, 25°, 30°, 33°, and 37°. Nine strains had UV-induced mutations that caused no phenotype in the presence of wild-type PRP40 but died in the absence of region 2. These were designated slf mutants 1–9 (Table 2).

A trans test was used to prove that the UV-induced mutations were in chromosomal genes rather than in the prp402 plasmid. The prp402 plasmid was lost from candidate synthetic lethal strains and a fresh copy of the same plasmid was reintroduced. As expected, all candidates still exhibited synthetic lethality after 5-FOA treatment (data not shown). In addition, a test for 5-FOA hypersensitivity was used to demonstrate that the synthetic lethality was not due to a mutation that made the cells more susceptible to this drug; transformation of a PRP40 TRP1 plasmid allowed all candidates to survive on 5-FOA.

Each candidate was then backcrossed to the unmutagenized parent and tetrads were dissected to ensure that the synthetic lethal phenotype resulted from a single mutation. The diploid strain was also used to determine if the synthetic lethal allele was recessive to its wild-type allele (Table 2). Finally, we placed the synthetic lethal mutants into complementation groups by mating them to each other and testing for synthetic lethality in the diploid strain on 5-FOA. Seven candidates, encompassing five different complementation groups, passed all tests (Table 2). Two complementation groups were dominant while the other three were recessive (Table 2).

Characterization and cloning of the slf mutants:
The slf mutants were evaluated for their phenotypes in combination with other alleles of prp40 (Table 2). Such allele specificity tests can reveal how the slf genes interact with PRP40. For example, an slf mutation that affects the stability or synthesis of Prp40p might be synthetically lethal with many prp40 alleles. In contrast, if the slf protein interacts specifically with one region of Prp40p, it would be expected to cause synthetic lethality only with prp40 alleles that affect that region. Each slf mutant was tested with the prp40-K4-A4 and prp40ARM alleles. prp40-K4-A4 has four lysines in region 2 changed to alanines, while prp40ARM has a 12-residue deletion of a region containing five arginines (Fig 1B). These tests defined at least four different allele specificity profiles among the seven slf strains (Table 2). Importantly, no slf mutant displayed synthetic lethality with all alleles of prp40 tested, indicative of specific interactions.

Suppressors of the recessive synthetic lethal mutations were cloned by complementation of the synthetic lethal phenotype by a CEN-based genomic library. Complementing plasmids were recovered, sequenced, and the individual open reading frames present were individually subcloned to determine which ones conferred complementation. In this way, we identified SME1, CRM1, and YNL187w as suppressors of the synthetic phenotypes caused by the three recessive synthetic lethal complementation groups. SME1 suppressed slf7, CRM1 suppressed slf4 and slf8, and YNL187w suppressed slf2 and slf9 (Table 2). We then sequenced the genomic copies of CRM1 from the slf4 and slf8 strains and YNL187w from the slf2 and slf9 strains to determine if they contained the synthetic mutations. ynl187w contained an early frameshift mutation at codon 55 (data not shown). Furthermore, deletion of ynl187w, in an otherwise wild-type background, conferred synthetic lethality with prp402, suggesting that slf2 and slf9 are ynl187w (data not shown). Interestingly, CRM1 contained no mutations in its coding sequence or in its promoter. Therefore, we conclude that mild overexpression of CRM1 confers dosage suppression of the slf4 and slf8 mutations. We did not sequence SME1 from slf7.

Sme1p is an essential splicing factor and a component of the yeast U1, U2, U4, and U5 snRNPs (BORDONNE and TARASSOV 1996 ). Crm1p is an essential nuclear export factor that recognizes leucine-rich NES sequences (FORNEROD et al. 1997A , FORNEROD et al. 1997B ; NEVILLE et al. 1997 ; STADE et al. 1997 ). Interestingly, YNL187w is a nonessential gene encoding a consensus leucine-rich NES sequence (Fig 1C and Fig 2C; GIAEVER et al. 2002 ). Therefore, two genes that have a link to nuclear export display genetic interactions with region 2 of Prp40p. In metazoan cells, using an adapter protein, Crm1p exports U snRNAs from the nucleus to the cytoplasm during snRNP biogenesis (FORNEROD et al. 1997A ; OHNO et al. 2000 ). Although we did not expect compartmentalized snRNP biogenesis to occur in yeast, we investigated whether region 2 of Prp40p was required for some nuclear export event.

View larger version (40K): In this window In a new window Download PPT slide   Figure 2. The NES sequences of PRP40 interact genetically with SLF4, YNL187w, and themselves. (A) Yeast strains with different combinations of PRP40 or prp40-nes1 and SLF4 or slf4 were tested for growth at 36° on YEPD plates. (B) Yeast strains with PRP40, prp40-nes1, prp40-nes2, or prp40-nes1/nes2 were tested for growth at 33° on YEPD (SLF4 was wild type in all cells). (C) Yeast strains with PRP40 or prp40-nes1 were tested for growth at 36° in the presence or absence of YNL187w. The genotypes are indicated around each plate.

Prp40p contains NES sequences:
Region 2 of Prp40p contains a leucine-rich sequence that fits the NES consensus sequence (Fig 1C). This sequence (274-L k e L r e y L n g I-284), called NES1, differs from the consensus (L x_(2-3) L x_(2-3) L x L/I; BOGERD et al. 1996) only in the spacing of the last two hydrophobic residues. Intriguingly, a second sequence (340-L q n k L n e L r L-349; NES2) that fits the NES consensus lies 58 residues downstream of region 2 in Prp40p (Fig 1C).

Since NES1 lies primarily within region 2, we tested whether the loss of NES1 was responsible for the growth defect of prp402. Conservative amino acid changes in the leucine residues of NES1 (Fig 1C) were made and their effect on growth was evaluated. As seen in Fig 2A, the prp40-nes1 mutant strain (prp40-nes1 SLF) displayed a growth defect relative to wild type (PRP40 SLF) at 36°. However, the loss of NES1 did not account for all of the function lost when region 2 was deleted, as prp40-nes1 grew better than prp402 (data not shown). Nonetheless, since the prp40-nes1 strain exhibited a growth defect at elevated temperatures, we conclude that NES1 is important for Prp40p function. Furthermore, we conclude that region 2 contains amino acids involved in a separate function.

The PRP40 NES sequences interact with SLF4 and YNL187w:
We next tested whether the loss of NES1 was responsible for the synthetic lethality observed when prp402 is combined with slf4. In the presence of wild-type PRP40, slf4 mutants grew at the same rate as wild-type cells at 36° (Fig 2A, compare PRP40 SLF4 and PRP40 slf4). As described above, the prp40-nes1 mutant strain displayed a growth defect at 36° (Fig 2A, prp40-nes1 SLF4). However, when the prp40-nes1 and slf4 mutations were combined in the same strain, a much stronger 36° growth defect resulted (Fig 2A, prp40-nes1 slf4). We note that this phenotype did not completely mimic the lethal phenotype of prp402 slf4, presumably because prp402 affects another function of Prp40p. Nonetheless, this synergistic growth defect demonstrates a functional interaction between SLF4 and the NES1 sequence of Prp40p. Furthermore, dosage suppression of the prp402 slf4 synthetic lethality by CRM1 suggests a functional link between the NES1 sequence and CRM1.

We also tested whether NES2 was important for Prp40p function. Conservative amino acid changes in the leucine residues of NES2 were made and their phenotypes were tested (Fig 1C). Surprisingly, strains with the prp40-nes2 mutation displayed no growth defect at any temperature. For example, growth at 33° is shown in Fig 2B. It is conceivable that the lack of a mutant phenotype after mutation of NES2 is due to the difference in severity of mutations between NES1 and NES2 (Fig 1C). However, mutation of two alanines is sufficient to disrupt the interaction of other NESs with Crm1p (WEN et al. 1995 ). The simplest explanation for this result is that NES1 and NES2 are functionally redundant. We tested this hypothesis by constructing the prp40-nes1/nes2 double mutant. This mutant allowed us to test for cis synthetic lethality between these two regions. When grown at 33°, no growth defect was observed when either NES was mutated singly (Fig 2B, prp40-nes1 and prp40-nes2). However, when mutations in both NESs were combined in cis, a strong growth defect at 33° resulted (Fig 2B, prp40-nes1/nes2). This synergistic defect suggests a partial functional redundancy between the two NES sequences. Although it is unknown whether these growth defects result from gross changes in protein structure, two observations suggest they do not. First, the leucine-to-alanine changes are conservative mutations not likely to disrupt global protein structure. Second, Western analysis showed that prp40p-GFP alleles were expressed at similar levels (data not shown).

If NES1 and NES2 were completely functionally redundant, mutation of NES1 would cause no phenotype as long as NES2 were wild type. However, mutation of NES1 did cause a significant growth defect at 36° (Fig 2A; compare PRP40 SLF4 and prp40-nes1 SLF4). We found no synthetic lethal interactions between prp40-nes2 and slf4 (data not shown). The simplest model to explain these functional interactions predicts that NES1 and NES2 act as independent and redundant export signals; mutation of either NES alone does not prevent export but mutation of both NES sequences does.

Since an allele of ynl187w was also synthetically lethal with prp402, and because Ynl187wp is predicted to also contain a leucine-rich NES sequence, we tested whether the NES sequences of PRP40 interacted genetically with YNL187w. As expected, deletion of ynl187w caused no growth defect in the presence of wild-type PRP40 (Fig 2C, PRP40 ynl187w; GIAEVER et al. 2002 ). As noted above, prp40-nes1 caused a growth defect at 36° in the presence of YNL187w (Fig 2A and Fig C). Interestingly, however, deletion of ynl187w in the prp40-nes1 strain exacerbated this phenotype (Fig 2C, prp40-nes1 ynl187w). Similarly, a triple mutant, in which ynl187w was deleted in the prp40-nes1/nes2 strain, displayed a growth defect more severe than any combination of two of these mutations (data not shown). These synergistic defects are suggestive of a functional interaction between ynl187w and the NES sequences of Prp40p. We have not, however, attempted to map the YNL187w region of functional interaction to the putative leucine-rich NES.

We next tested for a physical interaction between Prp40p and Crm1p, using both glutathione S-transferase pull downs and a yeast two-hybrid assay. In extensive trials, no specific interaction was observed. Although a weak interaction was detected by two-hybrid assay, it was not dependent on intact NES sequences. Although addition of U1 snRNA and Gsp1p to pull-down reactions had no effect, we believe that other components are likely required for a specific interaction between the NES sequences and Crm1p. Thus, we instead attempted to test the function of the NES sequences of Prp40p.

The Prp40p NES sequences are required for splicing:
The presence of NES sequences in the essential splicing factor PRP40 suggests that nuclear export of Prp40p, the U1 snRNP, or other splicing factors may be required for splicing. To test this, we utilized the temperature-sensitive phenotype caused by mutation of NES1 to perform an in vivo splicing assay. Cells were grown at a permissive temperature (30°) to midlog phase and then shifted to restrictive temperature (37°). Total RNA was isolated from the cells prior to and after temperature shift and the level of splicing of two RNAs was measured by primer extension at different intervals after temperature shift. Using a primer that annealed to the second exon of the intron-containing U3 RNA allowed us to distinguish unspliced RNA from spliced RNA (Fig 3A, lane A). The mature U3 RNA is transcribed and processed from two genes, each containing a different size intron that is removed by the spliceosome (MYSLINSKI et al. 1990 ). Thus, primer extension reveals two different-sized pre-U3 RNAs but only one mature U3 RNA (Fig 3A, lane A). Temperature shift caused no increase in unspliced RNA levels in a wild-type strain (Fig 3A, lanes A–E) but caused a dramatic increase in unspliced RNA levels in both the prp40-nes1 and the prp40-nes1/nes2 mutant strains (Fig 3A, lanes F–J and K–M, respectively). Such an increase in unspliced RNA is indicative of a reduction in splicing efficiency. Therefore, we quantified the RNA levels of each species by densitometry. The ratio of the amount of the shorter spliced species to the amount of the longer unspliced species yielded a relative measurement of the splicing efficiency for that sample. At permissive temperatures, both prp40-nes1 and prp40-nes1/nes2 mutations displayed similar splicing efficiencies as an isogenic wild-type strain (Fig 3B, open bars). After shift to 37°, however, both prp40-nes1 and prp40-nes1/nes2 mutations displayed significantly reduced splicing efficiencies (Fig 3B, prp40-nes1 and prp40-nes1/nes2, shaded and solid bars) while the splicing of an isogenic wild-type strain remained unchanged (Fig 3B, PRP40, shaded and solid bars). Similar results were observed for the splicing efficiency of RPL30 mRNA (data not shown). Mutation of the NES sequences mimicked the splicing defect observed in a strain expressing prp40-10, an allele containing a nonsense mutation just downstream of NES2 at W379 (Fig 3A, lanes N–P; Fig 3B, prp40-10; KAO and SILICIANO 1996 ). Thus, disruption of the NES1 sequence in Prp40p caused not only a temperature-sensitive growth defect but also a temperature-sensitive splicing defect. These data suggest that prp40-nes mutants die at elevated temperatures because they are defective at splicing their mRNAs. These data also suggest that nuclear export of Prp40p is required for pre-mRNA splicing. It is possible, however, that the leucine residues of the NES sequences perform auxiliary roles in the splicing reaction. The splicing defect resulting from mutation of the NESs therefore may result indirectly from the disruption of Prp40p export, directly from disruption of the splicing reaction, or from disruption of both.

View larger version (38K): In this window In a new window Download PPT slide   Figure 3. Mutation of the NES sequences of Prp40p caused a splicing defect in vivo. (A) Primer extension analysis revealed the levels of pre-U3 RNA and mature U3 RNA isolated from strains expressing wild-type or temperature-sensitive alleles of prp40 grown at 30° (0 hr) or shifted to 37° for 2, 4, 8, or 12 hr. The time points for each lane are denoted above the gel. The two top bands correspond to pre-U3 RNA arising from two independent genes differing in intron size (MYSLINSKI et al. 1990 ). A primer for the U5 snRNA was added as an internal control (lanes A–P). Lane Q is a control lane containing only the U3 RNA primer and lane R contains digested, 32P-labeled pBluescript as a size marker. The asterisk (*) denotes a band arising from nonspecific priming by the U5 snRNA primer as it does not appear in lane Q. (B) The relative splicing efficiency of different strains before and after temperature shift as calculated by dividing the total mature U3 RNA level by the total pre-U3 RNA level in each lane and dividing by 1000. Open bars, 30°; shaded bars, 2 hr at 37°; solid bars, 4 hr at 37°.

We further tested whether crm1 or ynl187w mutations also conferred splicing defects. However, the deletion of neither ynl187w nor a temperature-sensitive allele of crm1 (xpo1-1) conferred a splicing defect in vivo (data not shown; STADE et al. 1997 ).

The Prp40p NES sequences affect subcellular localization:
To test if the NES sequences of Prp40p function as true nuclear export signals, we fused the gene encoding green fluorescent protein (GFP) to the carboxy terminus of PRP40. We first expressed this fusion protein from its own promoter on a CEN-based plasmid. Unfortunately, expression was not high enough to visualize any Prp40p-GFP. We then constructed a 2-µm plasmid that expressed PRP40-GFP from the GPD promoter. This high expression construct allowed Prp40p-GFP to be visualized easily. Importantly, both the low-expression and high-expression constructs fully complemented a prp40 disruption (data not shown).

We investigated the localization of Prp40p-GFP by fluorescence microscopy and, as expected of an essential splicing factor, it was almost completely nuclear (Fig 4A). We conclude that any nuclear export of Prp40p by its NES sequences must be limited in some manner. Export may be a brief or rare event, it may involve only a small fraction of the cellular Prp40p pool, or it may lead to Prp40p's immediate degradation. In an attempt to uncouple Prp40p from this limiting factor or event, we deleted amino acids 1–256, leaving only the carboxy-terminal half of Prp40p fused to GFP. This construct, prp40256-GFP, contained both NES sequences and several putative nuclear localization sequences but lacked essential amino-terminal domains. This construct, therefore, could not complement a prp40 disruption but could be expressed in a wild-type background.

View larger version (62K): In this window In a new window Download PPT slide   Figure 4. Prp40p NES sequences function as nuclear export signals. (A) The subcellular localization of Prp40p fused to GFP is almost exclusively nuclear as determined by fluorescence microscopy. (B–E) The carboxy-terminal half of prp40 alleles was fused to GFP and overexpressed in wild-type cells. The subcellular localization of each protein was determined by fluorescence microscopy. A significant amount of cytoplasmic fluorescence was observed with constructs containing wild-type NESs (B) while constructs containing mutant NESs (C–E) were almost entirely nuclear. Shown to the right in B–E are DAPI fluorescence pictures of the same fields of view to locate nuclei. (F) The average ratio of the intensity of the nuclear to the cytoplasmic GFP in 50–100 cells. The variances between the NES mutants and the wild-type samples were compared by an F-test and the P values are shown.

By fluorescence microscopy, prp40256p-GFP was localized to the nucleus but unlike Prp40p-GFP, a significant amount of the protein was also detected in the cytoplasm (Fig 4B). This suggests that prp40256p-GFP was shuttling between the nucleus and the cytoplasm. The strong nuclear signal suggests that either import was more active than export or prp40256p-GFP was more stable in the nucleus. Mutation of the NES sequences in prp40256p-GFP caused a significant nuclear accumulation of the fusion protein, indicating that these sequences were required for nuclear export (Fig 4, compare B to C, D, and E). The intensity of GFP fluorescence in the nucleus and in the cytoplasm was calculated for 50–100 cells of each allele of prp40256-GFP. When the ratio of GFP detected in the nucleus to that detected in the cytoplasm was compared between alleles, the prp40256-nes1/nes2 double mutant had a twofold greater ratio than the prp40256 allele (Fig 4F). This difference was statistically significant (P = 3 x 10^-31) as determined by an F-test. Importantly, mutation of either NES1 or NES2 alone also caused a statistically significant nuclear accumulation (P = 4 x 10^-24 and P = 0.005, respectively) with NES1 mutations causing the most dramatic nuclear accumulation. These results clearly demonstrate that the NES sequences in prp40256p-GFP function in subcellular localization. Although it is possible that this shuttling occurs only with the carboxy-terminal half of Prp40p, we expect that the NES sequences also direct the export of full-length Prp40p because a significant amount of Prp40p sequence still surrounds the NESs in prp40256p-GFP.

prp40256p-GFP subcellular localization is affected by Crm1p:
Leptomycin B is a cytotoxin that specifically inhibits Crm1p function in many organisms including Xenopus laevis and S. pombe (NISHI et al. 1994 ; WOLFF et al. 1997 ). While S. cerevisiae Crm1p is resistant to this drug, a single-point mutation (creating a cysteine at amino acid 539) renders the S. cerevisiae protein leptomycin B sensitive (KUDO et al. 1999 ; NEVILLE and ROSBASH 1999 ). We visualized the subcellular distribution of prp40256p-GFP in a S. cerevisiae strain sensitive to leptomycin B (Lmb4). This strain displayed wild-type growth on rich media but died in the presence of 20–100 ng/ml leptomycin B (data not shown). Cells were grown to midlog phase prior to the addition of either carrier or 100 ng/ml leptomycin B for 45 min. Fluorescence microscopy revealed that leptomycin B caused a significant nuclear accumulation of prp40256p-GFP as compared to carrier alone (Fig 5, compare A with B). This difference is statistically significant as determined by an F-test (P = 6 x 10^-19; Fig 5C). As described in other instances, nuclear accumulation was observed as early as 15 min after cytotoxin addition (data not shown). Thus, inhibition of Crm1p mimicked the effect of NES mutations in prp40256p-GFP (compare Fig 5B with Fig 4E). As expected, no effect was observed upon the addition of leptomycin B to a wild-type CRM1, S. cerevisiae strain (data not shown). These results demonstrate that Crm1p function is necessary for the proper subcellular localization of prp40256p-GFP.

View larger version (55K): In this window In a new window Download PPT slide   Figure 5. Prp40256p-GFP localization requires Crm1p function. Threonine 539 of the S. cerevisiae CRM1 was mutated to cysteine, rendering it sensitive to leptomycin B (LMB; KUDO et al. 1999 ; NEVILLE and ROSBASH 1999 ). Cells were modified to express this LMB-sensitive allele as their sole source of Crm1p. (A) In the absence of LMB, but presence of carrier, the prp40256p-GFP fusion protein was distributed evenly between the nucleus and cytoplasm of these cells. (B) In the presence of 100 ng/ml LMB, a dramatic nuclear accumulation of prp40256p-GFP was observed. Shown to the right in A and B are DAPI fluorescence pictures of the same fields of view to locate nuclei. (C) The average ratio of the intensity of the nuclear to the cytoplasmic GFP in 50 cells of each sample, indicating the nuclear accumulation of prp40256p-GFP. The variances between the two samples were compared by an F-test and the P value is shown.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Here, we have performed a synthetic lethal screen in S. cerevisiae that identified a functional interaction between the essential splicing factor PRP40 and two genes displaying characteristics of nuclear export factors. One synthetic lethal gene encodes a putative leucine-rich NES. The synthetic phenotype caused by the other is suppressible by CRM1 overexpression. This synthetic lethality resulted from combining the aphenotypic slf mutations found in this screen with the slow-growing prp402 deletion. prp402 contains an internal deletion that removes a leucine-rich NES sequence fitting the consensus sequence recognized by Crm1p. Mutation of this NES sequence mimicked the internal deletion in conferring a synergistic defect when combined with slf mutations. Interestingly, Prp40p contains a second consensus NES sequence. Therefore, we have investigated the roles of these NES sequences in Prp40p function. We demonstrate that these NES sequences are functional for nuclear export through a Crm1p-mediated pathway. Moreover, these NES sequences are required for efficient pre-mRNA splicing.

The NES sequences of Prp40p:
Mutation of NES1 caused temperature-sensitive growth while the prp402 deletion caused slow growth at all temperatures. This indicated that another function disrupted in the prp402 allele contributed to the complete lethality found in our screen. Nonetheless, mutation of NES1 did cause a phenotype that was exacerbated by the aphenotypic slf4 mutation. Mutation of NES2 in Prp40p caused no detectable growth defect but did exacerbate the prp40-nes1 phenotype. A second gene synthetically lethal with prp402 is ynl187w, which also contains a leucine-rich NES sequence. The mutant phenotype of prp40-nes1 was also exacerbated by deletion of ynl187w. Similarly, mutation of both NES1 and NES2 in a ynl187w deletion strain caused a severe growth defect (worse than any combination of two of these mutations). However, the strain was still alive. These results suggest that NES1 of Prp40p is the most dominant export signal since mutation of NES2 alone or disruption of ynl187w alone caused no detectable growth defect. Unexpectedly, no combinations of NES mutations completely recapitulated the lethality observed between prp402 and the slf mutations.

Evidence for a second nuclear export pathway:
There appears to be some function associated with region 2 of PRP40 that is independent of NES1 and synthetically lethal with the slf mutations. It is conceivable that a second nuclear export factor recognizes other sequences of region 2 in a redundant export pathway. Recently, Brownawell and Macara characterized a novel nuclear export factor from Homo sapiens that they named Exportin-5. It exports dsRNA-binding proteins from the nucleus by directly binding to their dsRBDs (BROWNAWELL and MACARA 2002 ). The putative yeast homolog of Exportin-5 is MSN5 (BROWNAWELL and MACARA 2002 ), a nonessential nuclear export and import factor (KAFFMAN et al. 1998 ; YOSHIDA and BLOBEL 2001 ). Considering the similarity of region 2 to a dsRBD, it is tempting to speculate that Msn5p may recognize region 2 and aid Crm1p in exporting Prp40p. In this model, the synthetic lethality between prp402 and slf mutations would result from the loss of two redundant export pathways.

Brownawell and Macara further demonstrated that the binding of Exportin-5 or dsRNA to the dsRBDs in ILF3 was mutually exclusive (BROWNAWELL and MACARA 2002 ). In the absence of dsRNA, ILF3 was exported to the cytoplasm. Although no RNA-binding activity has been demonstrated with region 2 of Prp40p, it is plausible that Msn5p exports Prp40p that is not bound to dsRNA, i.e., the U1 snRNP. Similarly, the leucine-rich NES sequences of Prp40p may be inaccessible to Crm1p when bound to dsRNA. In this way, cells may export free Prp40p as a mechanism of protein turnover. This model predicts that Prp40p is less stable in the cytoplasm than in the nucleus and explains why, despite so many nuclear export signals, very little cytoplasmic Prp40p is ever visualized.

Synthetic lethality with ynl187w:
The prp402 allele also caused complete lethality when combined with a mutation in the nonessential gene, ynl187w. Interestingly, both PRP40 and YNL187w encode consensus NES sequences. The downstream NES of PRP40 is 70% identical to the NES in YNL187w (Fig 1C). The simplest explanation of these synthetic lethal results is that Prp40p and Ynl187wp contain redundant NES sequences, each of which is recognized independently by Crm1p. Mutation of both NESs (prp402 ynl187w-slf) would thus result in the loss of nuclear export of a complex. This model predicts that at least some Ynl187wp and Prp40p reside, at least transiently, in a common complex. In support of this, YNL187w was shown by yeast two-hybrid analysis to interact with Sm B', a component of the U1 snRNP (FROMONT-RACINE et al. 1997 ). These observations suggest that the U1 snRNP may be exported from the nucleus. However, Ynl187wp does not copurify with U1 snRNPs (GOTTSCHALK et al. 1998 ), co-immunoprecipitate with U1 snRNP factors (GAVIN et al. 2002 ), or copurify with spliceosomes (STEVENS et al. 2002 ). In contrast, Ynl187wp co-immunoprecipitates with a complex of cell-cycle-regulating, serine/threonine phosphatases (GAVIN et al. 2002 ). Therefore, Ynl187wp and Prp40p may be components of a different common complex or two independent, redundant complexes.

A role for Prp40p in exporting the U1 snRNP:
There are several plausible explanations for the temperature-sensitive phenotypes caused by mutation of Prp40p's NES sequences. For example, the mutations in the NES sequences could disrupt an essential interaction in a temperature-sensitive manner. This interaction may be with Crm1p or a splicing factor within the spliceosome. Alternatively, nuclear export may be required only at elevated temperatures. We prefer the latter explanation for two reasons. First, in other NES sequences, mutation of two leucines to alanines is sufficient to completely disrupt the interaction with Crm1p, regardless of temperature (WEN et al. 1995 ). Second, these are conservative mutations (leucine-alanine) and are not likely to disrupt the overall structure of prp40p and therefore should not disrupt other essential protein-protein interactions. In this model, a more stable U1 snRNP or spliceosome may be required at elevated temperatures and this may be achieved by nuclear export, modification, and reimport of Prp40p, the U1 snRNP, or some other complex. At lower temperatures, the U1 snRNP, or spliceosome, would be more stable and would not require the modification. In support of such a model, alternative forms of yeast snRNPs have been previously reported in response to heat shock (BRACKEN and BOND 1999 ). Thermotolerance of pre-mRNA splicing can be achieved by the addition of heat-shock proteins to snRNPs in many species (VOGEL et al. 1995 ; BRACKEN and BOND 1999 ). It is possible that this addition of heat-shock proteins to stabilize snRNPs is a cytoplasmic event and may require Prp40p export. We note that it is also possible that export is required for an entirely different reason such as snRNP recycling, disposal of unbound Prp40p or dysfunctional snRNPs, regulation of splicing, or export of mRNAs.

Although metazoan snRNPs are processed and assembled in the cytoplasm, a cytoplasmic modification of the yeast U1 snRNP is unexpected for several reasons (KOMEILI and O'SHEA 2001 ; KUERSTEN et al. 2001 ; WILL and LUHRMANN 2001 ). First, no snRNAs can be detected in the yeast cytoplasm (data not shown; C. GUTHRIE, personal communication). Second, cap hypermethylation and 3' end trimming of snRNAs, hallmarks of snRNP biogenesis, are nuclear restricted in yeast cells (VAN HOOF et al. 2000 ; MOUAIKEL et al. 2002 ). Third, there is no obvious homolog of the metazoan snRNP export and import factors, PHAX and Snurportin1, respectively (HUBER et al. 1998 ; OHNO et al. 2000 ). Last, all essential proteins of the yeast U1 snRNP appear to contain their own nuclear localization signals.

Despite these differences, it is conceivable that a minor modification of the U1 snRNP is required in the cytoplasm for efficient splicing at elevated temperatures. This may not be a U1 snRNP-specific pathway since one U5 snRNP protein, Snu114p, also contains a consensus NES sequence. However, no protein associated with the U2 or U4/U6 snRNPs appears to contain an NES sequence. In support of a U1 and U5 snRNP-specific export event, it is interesting to note that partially assembled U5 snRNPs always copurify with U1 snRNPs from yeast extracts, suggesting a common pathway of assembly or recycling (GOTTSCHALK et al. 2001 ). Importantly, this di-snRNP complex contains both Prp40p and Snu114p (GOTTSCHALK et al. 2001 ).

Prp40p NES sequences are functional:
We have shown that the NES sequences in Prp40p are required for proper pre-mRNA splicing. Mutation of NES1 or both NES1 and NES2 caused a defect in splicing at restrictive temperatures in vivo. Interestingly, this defect began to appear 30 min after temperature shift and became maximal by 2 hr (data not shown). This defect appeared too quickly to result from a loss of classical snRNP biogenesis and suggests a different function for Prp40p export. Similarly, temperature shift had no effect on U1 snRNA levels (data not shown). It is possible that the immediate splicing defect results from direct disruption of the splicing reaction in the presence of NES mutations. Alternatively, in the absence of nuclear export, the pool of U1 snRNPs (possibly missing a cytoplasmic modification) may be functional at lower temperatures but cannot function at higher temperatures. Such a situation would account for the immediate splicing defect occurring after temperature shift.

We have also shown that the NES sequences in Prp40p function as true nuclear export signals in the context of surrounding Prp40p sequence. Deletion of the amino-terminal half of Prp40p redistributed the protein from almost entirely nuclear to nuclear and cytoplasmic. Mutation of the NES sequences redistributed it back to almost exclusively nuclear. We envision that the carboxy-terminal half of Prp40p-GFP shuttled between the nucleus and the cytoplasm using intact nuclear localization signals and the NES1 and NES2 sequences. This is supported by the presence of several regions of basic residues present in prp40256. Mutation of the NES sequences disrupted export while not affecting import, resulting in nuclear accumulation. Addition of the Crm1p inhibitor LMB to cells expressing the shuttling carboxy-terminal half of prp40p mimicked the nuclear accumulation observed by mutating the NES sequences. Thus, disruption of either Crm1p function or the NES sequences of prp40p resulted in nuclear accumulation. These experiments demonstrate that the NES sequences in Prp40p function as true nuclear export signals.

Although these experiments visualized overexpressed prp40p-GFP alleles, we believe that the distribution was representative of endogenous Prp40p for several reasons. Wild-type Prp40p fused to GFP fully complemented a prp40 null strain. Furthermore, Prp40p-GFP was almost exclusively nuclear as expected of a splicing factor. Finally, all calculations of nucleus/cytoplasm ratios were calculated from a diverse set of cells expressing different levels of fusion protein and extremely high-expressing cells were omitted from calculations.

Together our results suggest a model in which Prp40p and Ynl187wp contain redundant, functional leucine-rich nuclear export signals that aid in a Crm1p-mediated nuclear export event. Moreover, our data suggest that this export is necessary for efficient splicing of pre-mRNAs at elevated temperatures. The most obvious candidate cargo for Prp40p and Ynl187wp to export is the U1 snRNP. Several reasons for its export can be hypothesized including a cytoplasmic modification during snRNP biogenesis, a snRNP-recycling event, a regulatory mechanism, an mRNA trafficking mechanism, or a protein turnover mechanism. It is also possible that Prp40p contains a second region responsible for export via a different export pathway, such as the Msn5p pathway. It will be interesting to address the requirements of region 2 and MSN5 in future experiments. Discovery of the function of Prp40p export will be paramount.

	FOOTNOTES

¹ These authors contributed equally to this work.
² Present address: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455.

	ACKNOWLEDGMENTS

We are grateful to Minoru Yoshida for generously providing leptomycin B. We also thank Erin Asleson, Julius SternJohn, Keiko Sakai, Maki Inada, and Christine Guthrie for helpful discussions and Erin Asleson for comments on the manuscript. This work was supported by National Institutes of Health grant GM 44628 and a 3M graduate fellowship to B.L.O.

Manuscript received March 19, 2003; Accepted for publication September 22, 2003.

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