Nuclear pore proteins are involved in the biogenesis of ... - Europe PMC

The EMBO Journal vol.15 no.9 pp.2270-2284, 1996

Nuclear pore proteins of functional tRNA

are

involved in the biogenesis

George Simos, Hildegard Tekottel, Henri Grosjean2, Alexandra Segref, Kishore Sharma', David Tollervey' and Eduard C.Hurt3 University of Heidelberg, Institut fur Biochemie I, Im Neuenheimer Feld 328, D-69120 Heidelberg, 'EMBL, Meyerhofstrasse 1, D-69117, Heidelberg, Germany and 2CNRS, Laboratoire d'Enzymologie, F-91198 Gif-sur-Yvette, France

3Corresponding author

Loslp and Puslp, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsplp. LOS], PUS] and NSPI interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Puslp is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Loslp was shown previously to be required for efficient pre-tRNA splicing; we report here that Loslp localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Puslp and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the losilpusil double mutant, as well as several thermosensitive nucleoporin mutants including nspl, nupll6, nupl33 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA. Keywords: nuclear pore complex/nucleocytoplasmic transport/nucleoporin/pseudouridine synthase/tRNA

Introduction Nucleocytoplasmic transport which is energy dependent and carrier mediated takes place through the nuclear pore complexes (NPCs) (Davis, 1995). A lot of information has become available recently concerning the nuclear import of proteins (reviewed in Melchior and Gerace, 1995; Simos and Hurt, 1995) but the mechanism of nuclear export of RNA is less well understood. For their export, different classes of RNA depend on specific signals which could either be part of the RNA moiety or provided by the protein components of the ribonucleoprotein particles (for review see Izaurralde and Mattaj, 1995). Recent studies have identified a nuclear export sequence (NES) in proteins that are exported actively from the nucleus, such as rev and PKI (Fischer et al., 1995; Gerace, 1995; Wen et al., 1995). RNA species may also be exported from the nucleus by associating with NES-containing proteins. This appears to be true for a subset of RNAs,

such as viral mRNAs containing an RRE (Rev responsive element), snRNAs and SS RNA (Fischer et al., 1995). Both nuclear protein import and RNA export require a functional Ran GTPase cycle. Components of this cycle such as the small nuclear GTPase Ran, the cytoplasmic protein RanGAP (RanGTPase-activating protein or Rnalp) and the nuclear Ran-specific nucleotide exchange factor RCC 1 have been implicated in both aspects of nucleocytoplasmic transport, in vitro and in vivo (Becker et al., 1995; Bischoff et al., 1995; Cheng et al., 1995; Corbett et al., 1995). Nuclear pore proteins also play an active role in both nuclear protein import and RNA export (reviewed in Doye and Hurt, 1995; Melchior and Gerace, 1995; Simos and Hurt, 1995). In yeast, nucleoporins Nsplp, Nup49p, Nup57p and Nic96p form a nuclear pore subcomplex which is required for protein uptake by the nucleus (Grandi et al., 1995b). A separate pool of Nsplp interacts physically with the essential nucleoporin Nup82p, depletion of which causes a defect in poly(A)+ RNA export but not protein import (Grandi et al., 1995a). These results suggest that Nsplp may participate in different nucleocytoplasmic transport processes via multiple interactions with other nucleoporins. Nuclear poly(A)+ RNA accumulation was also observed for a number of other nucleoporin mutants including Nuplp (Schlaich and Hurt, 1995), Nup133p/Rat3p (Doye et al., 1994; Li et al., 1995), Nup l 59p/Rat7p (Gorsch et al., 1995), Nup 16p and Nupl145 (Fabre et al., 1994). Another group of nucleoporins, Nup84p, Nup85p and Nupl2Op, are physically associated and are also required for efficient nuclear export of poly(A)+ RNA (Siniossoglou et al., 1996). All this shows that nuclear pore proteins are required for mRNA export. However, the absence of suitable test systems in yeast has made it difficult to determine whether the export of other classes of RNA, such as rRNA and tRNA, is also affected in the nucleoporin mutants. tRNA molecules are synthesized by RNA polymerase III as precursors which undergo a complex maturation pathway that includes trimming of the 5' and 3' ends, addition of three terminal CCA residues and modification of a number of nucleosides (Hopper and Martin, 1992). Furthermore, in the yeast Saccharomyces cerevisiae, ~20% of the genes encoding tRNAs contain introns which are removed from pre-tRNAs by a tRNA-specific splicing machinery (Culbertson and Winey, 1989). Base modifications are added in a stepwise manner and most of them are introduced prior to the removal of the intron (Nishikura and De Robertis, 1981). For some modifications in the anticodon the presence of the intron is a strict requirement (Johnson and Abelson, 1983; Strobel and Abelson, 1986; Szweykowska-Kulinska et al., 1994). Removal of introns from pre-tRNAs in yeast involves a pre-tRNA splicing endonuclease, a tRNA ligase and an NAD-dependent 2'-

27© Oxford University Press 2270

Nuclear pore proteins and biogenesis of tRNA

phosphotransferase (Westaway and Abelson, 1995). The splicing reaction probably takes place at the nuclear envelope since the endonuclease is a membrane protein (Peebles et al., 1983) and the tRNA ligase is localized preferentially on the inner side of the nuclear membrane and close to the NPC (Clark and Abelson, 1987). A tRNA modification enzyme, Trmlp, a N2,N2-dimethylguanosine methyltransferase, was also localized to the inner nuclear membrane (Rose et al., 1995). Export of tRNA from Xenopus oocyte nuclei was shown to be carrier mediated (Zasloff, 1983). Mutations in highly conserved regions of tRNA inhibited the processing of precursor transcripts as well as nuclear export of the mature product, suggesting that the structure of tRNA is important for both maturation and export (Tobian et al., 1985). Furthermore, unspliced or partially processed pretRNAs are discriminated by the export machinery since they cannot exit from the nucleus (Haselbeck and Greer, 1993). tRNA export differs in some aspects from the export of other RNA species, i.e. (i) export of microinjected tRNA from Xenopus oocyte nuclei exhibits faster kinetics than other RNA classes (Jarmolowski et al., 1994), (ii) homopolymeric RNAs inhibit the export of 5S RNA, mRNA and U snRNAs, but not of tRNA (Jarmolowski et al., 1994) and (iii) tRNA export apparently was not affected in a mammalian RCC 1 mutant whereas nuclear export of mRNA and snRNA is inhibited (Cheng et al., 1995). This suggests that the tRNA export mechanism may be functionally distinct from those regulating export of other RNAs. A number of nucleoporin mutants are defective in the splicing of pre-tRNAs (Sharma et al., 1996), indicating that pre-tRNA splicing and nuclearcytoplasmic transport are somehow coupled. A number of genetic screens were devised in yeast to identify trans-acting components involved in tRNA biogenesis. This approach identified several tRNA splicing and modification enzymes as well as proteins of unkown biochemical function (Hopper, 1990). These include the LOS] gene which was identified as a mutant exhibiting conditional loss of tRNATYr suppressor activity (Hopper et al., 1980). Accordingly, los] mutants produce reduced levels of functional suppressor tRNAs and accumulate endtrimmed, but unspliced pre-tRNA although they apparently contain normal levels of tRNA splicing endonuclease and ligase activities (Hurt et al., 1987; Shen et al., 1993). In this work, we have identified a functional interaction between the nuclear pore protein Nsplp, Loslp, which is also localized at the NPC, and a novel protein, Puslp, which is the first eukaryotic tRNA pseudouridine synthase characterized so far. Since suppressor tRNA activity is not only impaired in los] mutants, but also in several other nucleoporin mutants, it is clear that nucleoporins are required for the normal synthesis of mature, cytoplasmic tRNAs.

allele (Wimmer et al., 1992) (Figure lA, upper panel). In one case, the complementing activity was localized to an open reading frame (ORF) encoding a protein of 1100 amino acids which is identical to Loslp. The second complementing activity was localized to a novel ORF, potentially encoding a protein of 544 amino acids, which was called Pus Ip for pseudouridine synthase (previously referred to as Los2p, Sharma et al., 1996). The Puslp amino acid sequence is 43% identical over a stretch of 390 amino acids to an uncharacterized ORF adjacent to the yeast pyruvate carboxylase gene (Lim et al., 1988), which we tentatively named Pus2p (Figure iB). Furthermore, both Pus Ip and Pus2p are significantly homologous to the Escherichia coli tRNA pseudouridine synthase I (PSU-I, Kammen et al., 1988) and the yeast uncharacterized protein Deglp (Agostini Carbone et al., 1991) (Figure IB).

Results

Functional interactions between NSP1, LOS1 and PUS1 The fact that the sl mutant which allowed cloning of both LOS] and PUS] is complemented, not only by NSP] itself, but also by two different genes could be due to the presence of two independent mutations in both the LOS] and PUS] genes. This was investigated genetically by mating strain s1370 to a tester strain TF4 (see also Table I). Upon tetrad analysis, the segregation pattern of the synthetic lethal phenotype in haploid progeny was indicative of two unlinked mutations (see also Materials and methods). Like the starting mutant s1370, haploid progeny with the synthetic lethal phenotype were complemented by the NSPI, LOSI and PUS] genes (Figure lA, lower panel). An independent proof that these three genes functionally interact is to show that a synthetic lethal phenotype also can be generated by combining defined mutant alleles of nspl, los] and pusl (Figure 2). Gene disruption of PUS] (see Materials and methods) showed the gene to be nonessential (Figure 2A). LOS] is also not essential (Hurt et al., 1987) and single disruption mutants grow well at 30 and 37°C (Figure 2A). We therefore made various combinations of losl-, pusl- and nsp] mutant alleles. All double mutants losl- nspl]s, pusl- nspl]s (data not shown) and losl- pusl- are viable at 30°C, although the combination losl- pusl- exhibits impaired cell growth at 30°C and is not viable at 37°C (Figure 2A and B). This shows that LOS] and PUS] have a strong genetic interaction. Interestingly, yeast strains which have all three mutations in combination, losl-/pusl-/nspls, are not viable even at 30°C, which can be seen by the fact that a strain containing these three mutant alleles plus a wild-type NSPI gene on an URA3-containing plasmid cannot grow on 5-fluoroorotic acid (FOA)-containing plates (Figure 2B). This synthetic lethal phenotype can be complemented when the triple mutant is transformed with any of the three wildtype genes, LOS], PUS] (data not shown) or NSPI (Figure 2B). Thus, Loslp and PusIp both genetically interact with the nuclear pore protein Nsplp.

Components involved in tRNA biogenesis are found in a genetic screen for Nsplp-interacting components Two genes were identified, either of which independently can rescue the lethal phenotype of a synthetic lethal mutant (s1370) derived from a screen with a thermosensitive nsp]

Intracellular location of Loslp and Puslp Genetic interaction between NSPI, LOS] and PUS] could be indicative of a co-localization of the corresponding proteins at the NPC. The subcellular localization of Loslp and Puslp was analyzed, therefore, by indirect 2271

G.Simos et al.

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Fig. 8. Complementation of a synthetic lethal mutant of losi by TFC4. (A) Mutant s13 derived from the losi sl screen was complemented by a genomic DNA fragment containing the TFC4 gene (see Materials and methods). Both red/white sectoring (data not shown) and growth on 5-FOA plates of the sl 3 mutant were restored by a 4 kb Xmnnl restriction fragment which contains the TFC4 gene (two individual transformants), as well as by pUN100-LOSI. but not by the pUN100 vector alone. It was grown for 4 days at 30°C before the picture was taken. (B) The mutant s13 is impaired in growth at 37°C due to a mutation within TFC4 (tfc4-3). Equivalent amounts of cells (either undiluted or diluted in 1/10 steps) from the s13 mutant transformed with either pUNIO0 alone (vector), pUN100-LOSI or pUN100-TFC4. respectively, were spotted onto YPD plates and incubated at 30 or 37°C for 3 days. (C) The s13 mutant shows reduced levels of tRNA expression and is impaired in 5' and 3' pre-tRNA processing due to mutation in TFC4. RNA was extracted from [3H]uracil-labeled s13 (lanes I and 3) or s13 complemented by TFC4 (lanes 2 and 4) cells grown at 30°C and analyzed on a 6% urea-polyacrylamide gel. An equal amount of counts was loaded on each lane. Lanes I and 2: staining by ethidium bromide, lanes 3 and 4: fluorography. The positions of 25S and 18S (rRNA), 5.8S, 5S and tRNA are marked by bars. The arrow indicates higher molecular weight forms of tRNA present in the s13 mutant but absent from the TFC4-complemented strain.

Discussion Synthetic lethality allows dissection of distinct steps in tRNA biogenesis The genetic concept of synthetic lethality has allowed the identification of a number of components which interact in vivo with the yeast nuclear pore protein Nsplp. These include both nucleoporins which physically interact with Nsplp, and nucleoporins that functionally overlap with Nsplp without forming a biochemically stable complex (for review, see Doye and Hurt, 1995). Thus, Nspl functionally interacts with many NPC components. Here, a network of genetic interaction was found between Nsp p and components involved in tRNA biogenesis, including another NPC protein Los Ip, which was shown previously to be required for in vivo pre-tRNA splicing (Shen et al., 1993) and a novel intranuclear pseudouridine synthase Puslp. Furthermore, LOS] by itself is linked further to components of the tRNA biogenesis pathway, e.g. to Tfc4p (a transcription factor for tRNA genes), and a novel cytosolic protein involved in tRNA aminoacylation (G.Simos, unpublished results). All together, this shows that the combination of mutations affecting different steps in tRNA biogenesis such as transcription, modification, splicing, 5'/3' end processing, transport and aminoacyl-

ation, can give rise to synthetic lethal phenotypes, most likely due to synergism. One important impact of this finding is that synthetic lethality allows the tRNA biogenesis pathway to be dissected genetically, even though a mutation in one distinct step does not reveal an obvious defect in growth.

Pusip is a novel tRNA pseudouridine synthase Of the components which interact genetically with NSPI and LOS], Puslp was identified as a tRNA-modifying enzyme which converts one or several uridines into Ts at selected positions within specific tRNAs. Puslp is thus the first eukaryotic pseudouridine synthase to be cloned and characterized. Four prokaryotic pseudouridine synthases have been identified so far: (i) tRNA pseudouridine synthase I (Psul) from E.coli which forms ' at positions 38, 39 and 40 (Kammen et al., 1988); (ii) a P55 synthase specific for tRNA (Nurse et al., 1995); (iii) a rRNAspecific enzyme forming T516 in 16S RNA (Wrzesinski et al., 1995a); and (iv) a pseudouridine synthase able to form P746 in 23S RNA as well as T32 in tRNA (Wrzesinski et al., 1995b). Interestingly, Puslp and two as yet uncharacterized yeast proteins, Deglp and Pus2p, share significant homology to E.coli Psul, but not to the 2279

G.Simos et al.

other E.coli enzymes. In yeast, ' occurs in 15 different positions in the 34 cytoplasmic tRNAs sequenced so far (Grosjean et al., 1995). Distinct protein fractions catalyze the formation of TP13, P32, '55 (Samuelsson and Olsson, 1990) and '34/35/36 in tRNA (Szweykowska-Kulinska et al., 1994), suggesting that ' modification is mediated by different pseudouridine synthases which may act in a site-specific way. Moreover, a single tRNA pseudouridine synthase can modify several tRNA species harboring very different nucleotide sequences, suggesting that it may recognize a particular structure rather than a specific sequence (Bjork, 1995; Grosjean et al., 1996). In the case of '34 and T36, the determinants for the modifying enzyme reside essentially in the 60 bp long intron of yeast minor tRNAIIe (Szweykowska-Kulinska et al., 1994). This intron-dependent pseudouridine synthase activity evidently corresponds to Pus Ip. Our results also demonstrate that a single enzyme, Pus Ip, is responsible for formation of not only '34, T35 and '36 in the intron-containing tRNAIle, but also '27, thus exhibiting multisite specificity. Most of the modifications within cytoplasmic tRNAs occur before nuclear tRNA export (Nishikura and De Robertis, 1981). This is consistent with our findings that Puslp is localized inside the nucleus and acts before the removal of the intron in the case of tRNAIle. The role of nucleoside modification in tRNA is poorly understood. Modifications at or near the anticodon have been shown to be important for the efficiency of aminoacylation or codon recognition (Bjork, 1995). In the case of the yeast ochre suppressor tRNATYr (SUP6), the absence of ' at position 35 within the anticodon (as a result of the deletion of the intron), led to reduced suppressor tRNA activity (Johnson and Abelson, 1983). Puslp may actually be responsible for this modification, as it forms '35 in the isoacceptor tRNAIle which harbors an ochre UUA anticodon. Also, loss of function of the ochre suppressor tRNATYr SUP]], although not evident in the pusl- single mutant, was enhanced synergistically in the double pusllosl- mutant. Since Puslp is linked functionally to components of the NPC, tRNA modification appears to be an important requirement for the successive steps in tRNA biogenesis such as splicing and/or nuclear export. Alternatively, the observed synthetic lethality between NSPI, LOS] and PUS] could be an additive result of lack of tRNA modification, inefficient splicing and reduced nuclear export. Clearly, the in vivo analysis of tRNA modification enzymes, which often are not essential for cell growth (reviewed in Hopper and Martin, 1992), may be facilitated if perfomed in a genetic background in which their function becomes essential.

Several nucleoporins are required for the biogenesis of tRNA Loslp does not play a direct role in tRNA splicing, although los] mutants accumulate intron-containing tRNAs and exhibit loss of suppressor tRNA activity at an elevated temperature (Hurt et al., 1987; Shen et al., 1993). There can be at least two scenarios for how Loslp can affect tRNA splicing. As the tRNA ligase which is an essential component of the tRNA splicing machinery is localized close to the nuclear pores (Clark and Abelson, 1987) and the tRNA endonuclease appears to be an integral nuclear membrane protein (Peebles et al., 1983),

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processing of pre-tRNA may be coupled to the translocation step through the NPC (Culbertson and Winey, 1989). It is conceivable, therefore, that removal of Loslp from the nuclear pore may impair interaction of the tRNA splicing machinery with the NPC, thereby partially inhibiting the splicing reaction. Alternatively, Loslp may be involved directly in tRNA export reactions. Although we cannot address this point directly by experimental means (no tRNA export assay is available in yeast), los] mutants may accumulate mature tRNA inside the nucleus which could feed back on the splicing reaction, e.g. by end product inhibition. Interesting in this context is that the tRNA ligase is inhibited significantly by mature tRNA (Peebles et al., 1979). Furthermore, the mal mutant which accumulates unspliced RNA precursors including pretRNA (Hopper et al., 1978) may exhibit also a defect in nuclear export of tRNA. Rnalp was shown to be a cytoplasmic protein (Hopper et al., 1990) and was identified recently as the RanGAP (GTPase-activating protein) for Ran, an essential factor involved in nucleocytoplasmic transport (Becker et al., 1995). On the other hand, in mammalian cells, tRNA export apparently is not affected in a RCC1 mutant whereas nuclear export of mRNA and snRNA is inhibited (Cheng et al., 1995). Thus, it still remains open as to whether the Ran/RCC1/RNAl system is involved directly in nuclear tRNA export. When tRNA splicing was examined in other nucleoporin mutants, several of them accumulated unspliced tRNAs (e.g. Nsplp, Nup133, Nup49p, Nupll6p) while rRNA processing and mRNA splicing were not affected; this suggested a functional link between splicing and nuclear export of tRNA (Sharma et al., 1996). We have shown here that the function of a suppressor tRNA is also impaired in several nucleoporin mutants that include Nsplp, Nupl33p, Nupll6p, Nup84p, Nup85p and Nupl20p, but not NuplOOp, Nuplp and Nup57p. The data on loss of suppression do not correlate strictly with the data for the accumulation of unspliced pre-tRNA since the nup85::HIS3 partial disruption mutant does not accumulate unspliced tRNA (Sharma et al., 1996), yet exhibits a strong loss of suppressor tRNA activity (this work). Accordingly, Nup85p only affects the formation of functional suppressor tRNA, but not tRNA splicing. Most of the nucleoporin mutants which show loss of suppressor tRNA activity are also impaired in poly(A)+ RNA export (Doye et al., 1994; Fabre et al., 1994; Siniossoglou et al., 1996); therefore, these nuclear pore proteins may participate in a common step of nuclear RNA export. Loslp differs in this respect, since los] mutants are not impaired in either poly(A)+ RNA export or nuclear protein import, suggesting a more specific role in tRNA biogenesis. In any case, our data have shown that loss of suppressor tRNA activity and/or tRNA precursor accumulation is a phenotype associated with several nucleoporin mutants. Accordingly, genetic screens based on such a mutant phenotype may allow the identification of novel nucleoporins involved in the formation of functional tRNA. On the same lines, genes that have been found already on the basis of similar screens, but with their precise function and localization of their products unknown (e.g. PTA]; O'Connor and Peebles, 1992), may also encode nuclear pore proteins. The function of these genes may overlap with LOS], explaining its non-essentiality.

Nuclear pore proteins and biogenesis of tRNA

Material and methods Yeast strains, media and microbiological techniques The yeast strains used in this study are listed in Table I. Yeasts were grown on minimal SDC and rich YPD medium, sporulation of diploid cells on YPA plates and tetrad analysis w'ere performed according to (Wimmer et al., 1992). Minimal SDC medium/plates were supplemented by all amino acids and nutrients except the ones used for the selection or those which, if indicated, contained 5-FOA (CSM medium. BIOlO0. La Jolla. CA). In the case of the SDC -trp/low' ade plates. 2 ,tg/ml adenine per ml of medium w'as added. Genetic manipulations were performed as described (Sherman. 1990).

Plasmids The following yeast plasmids were used: pUNIOO, ARS/CEN plasmid with the LEU2 marker (Elledge and Davis, 1988): pUN20, ARS/CEN plasmid with the TRPI marker, the NOPI gene and the SUP]] which encodes a tyrosine-inserting ochre suppressor tRNA (Elledge and Davis, 1988): pCHl 122, YCp5O derivative (ARS/CEN 4) with the URA3 and ADE3 (Kranz and Holm. 1990): pRS414-ts nspl (L>S) (Wimmer et al.. 1992). Plasmids harboring yeast tRNAASP, minor tRNA"e (with or w'ithout an intron), yeast tRNAAIU and yeast tRNAPhe were described previously (Szweykowska-Kulinska et atl. 1994; Grosjean et al., 1996).

Cloning and sequencing of the LOS1 and PUS?

genes

The isolation and initial characterization of the synthetic lethal mutant of ts nspl. s1370, was described previously (Wimmer et al., 1992). For cloning of the gene(s) causing synthetic lethality in s1370, a yeast 1993) in a pUN100-LEU2 plasmid was genomic library (Jansen et transformed into s1370 by electroporation (Wimmer et al., 1992) and LEU+ transformants were first selected on SDC (-leu -trp) plates, before replica transfer on SDC (-leu -trp) and finally on YPD plates. Transformants which exhibited red/w'hite colony sectoring and could grow on FOA plates were identified and their pUNIOO plasmids were recovered (Wimmer et al., 1992). Six of these transformants contained pUN 100 plasmids with genomic inserts (in the range 8-12 kb) not allelic to NSPI which, if retransformed into s1370, could complement synthetic lethality. DNA restriction analysis revealed that the inserts were derived from two different genes. and a representative plasmid from each group. pUNI00-370-40 (containing the LOS] gene) and pUN100-37031 (containing the PUS] gene), was analyzed further. By subcloning appropriate restriction fragments in pUN 100. the smallest complementing region was determined. Finally, this DNA was sequenced essentially by primer walking with the dideoxy sequencing method (Sanger et al., 1977). Comparison of the LOS] DNA sequence determined in our laboratory w'ith the recently published LOSI DNA (Shen et al., 1993) show'ed that the sequences are 100% identical (data not shown). at.,

Gene disruption of LOS1 and PUS1 Disruption of the LOS] and PUS] genes was done by the one-step gene replacement method (Rothstein, 1983). In this study. the LOS] gene was disrupted by inserting a 0.9 kb long blunt-ended BamHI fragment containing the HIS3 gene into the EcoRV site of the LOS] ORF (Figure IA). It was shown earlier by disruption of almost the entire ORF (PvuII deletion) that LOS] is not essential for cell growth (Hurt et al.. 1987). The PUS] gene was disrupted by inserting the blunt-ended HIS3 gene into the NdeI site of the PUSI ORF (see also Figure IA). The disrupted genes were excised and the linear fragments used to transform the diploid strain RS453. HIS' transformants with the correct integration of the interrupted genes at the LOS] or PUS] locus. respectively, were verified by Southern analysis (data not shown). Correct integrants were sporulated and tetrads were dissected. A 4:0 segregation for viability and a 2:2 segregation for the HIS marker were found for the los] and pus] gene disruptions, showing that both genes are not essential for cell grow'th.

Construction of synthetic lethal mutants by combining losl, and ts nsp 1 mutant alleles

To construct a haploid yeast strain in which the disrupted losi and pllsI genes and a thermosensitive nspl allele are combined, a piusBmutant harboring pURA3-NSPI was mated to the double mutant loslnsppl". The resulting heterozygous diploids (called G3XB2) were sporulated and tetrad analysis was performed. For complete tetrads in which the HIS+/his- genotype segregates 2:2. one can predict that the two HIS+ progeny are losl::H153/pusl::HIS3/nspl::HIS3. A complete tetrad showing this segregation pattern was analyzed in greater detail for the segregation of the HIS, URA and LEU markers by plating cells on SDC -his, SDC -ura and SDC -leu plates, respectively. Two progenies are shown in Figure 3B. The HIS+ progeny losl::H1S3/pusl::HIS3/ nspl::HIS3 also contained plasmids pURA3-NSPl and pLEU2-ts nspl; therefore, we could test whether the triple combination losl-/pusl-/ nspl" gives synthetic lethality by plating this strain on FOA-containing plates at 30°C (Figure 3B). Synthetic lethality, however, was complemented by the presence of pLEU2-LOS 1 and pLEU2-PUS 1, respectively (data not shown).

Isolation of synthetic lethal (si) mutants of the losl::HlS3

allele To construct a losl::HIS3 strain with the ade2/ade3 markers which are required for the red/white colony sectoring assay (Wimmer et al., 1992). the losl- strain was mated to CH1462 (see Table I). The derived diploid strain was sporulated, and haploid progeny with both losl::H153 and ade2/ade3 were selected after tetrad analysis and transformed with pHT4467-URA3-ADE3-LOSl. The resulting screening strain for losl synthetic lethals which was available in both mating types (see Table I) formed red/white sectoring colonies on YPD plates (Wimmer et al.. 1992). UV mutagenesis of the sl screening strain wvas carried out essentially as described (Wimmer et al., 1992; Grandi et al., 1995b), and 20 000 surviving colonies were screened for a stable red, nonsectoring phenotype at 30°C and no growth on 5-FOA plates. Putative sl mutants were transformed finally with pUN1OO-LEU2-LOSl, and five of them regained a red/white sectoring phenotype and could grow' on 5FOA-containing plates. One of the five sl mutants, S13-BI (which was derived from the s13 strain after mating to a wild-type haploid RS453, followed by tetrad analysis and selection for haploid progeny exhibiting the sl-phenotype: see Table I), was transformed with a yeast genomic library inserted in pUN100 (described in Wimmer et al., 1992), and transformants showing a red/white sectoring phenotype and growth on 5-FOA-containing plates could be obtained. A complementing plasmid. which did not contain LOS], was recovered, and subfragments covering the entire complementing activity were sucloned into pUN100 for DNA sequence analysis. It turned out that the complementing activity of this plasmid is retained within a 4 kb long Xmnl fragment which contains the TFC4 gene (Marck et al., 1993). The accession number of TFC4 is L12722.

Construction of Losip and Puslp fusion proteins carrying protein A or myc as tag Epitope tagging of Loslp was done by fusing two IgG binding units from Staphylococcus aureus protein A to the N-terminal end of Loslp. For this gene fusion, a new PstI restriction site was aenerated at the ATG codon of LOS] by PCR-mediated mutagenesis in order to join the ORF with the SacI-PstI restriction fragment corresponding to two IgG binding units plus the NOPI promoter. The full-length ProtA-LOSI fusion gene was then subcloned in plasmid pUN100. For myc tagging of Loslp, a DNA fragment containing three myc epitopes in tandem (S.Kron, Whitehead Institute, Cambridge, MA: unpublished data) was cloned under the control of the NOPI promoter in pUN100. A SaclPstI fragment comprising the NOPI promoter (pNOPI) and the triple myc epitope was excised and the full-length (Myc)_-LOS1 fusion gene under the control of the NOPI promoter was constructed and subcloned in plasmid pUNI00 as above. Epitope tagging of Puslp with two IgG binding units from S.aureus protein A to the N-terminal end was done in a similar way. ProtA-Loslp/Puslp and Myc-Loslp were functional since they could complement the synthetic lethal phenotype of s1370 and the ts phenotype of strain losl- plisl- (data not show'n).

pus?

A haploid progeny derived from s1370 (called s1370-7) was obtained by mating the initial strain s1370 to a tester strain TF4 and selection for diploids on a SDC (-trp -leu) plate (see also Table I). Diploid cells w'ere sporulated and analyzed for the segregation pattern of the s1370 phenotype. Synthetic lethality did not segregate 2:2 (indicative of a single mutation), but 1:3 (synthetic lethal versus viable) and, in fewer cases. 0:4 and 2:2 (data not show'n). The observed pattern is indicative of two unlinked mutations in the LOS] and PUS] genes.

Expression of Loslp and Puslp in E.coli, purification of recombinant proteins and generation of antibodies The LOS] and PUS] ORFs were amplified by PCR using two primers that created a XhoI restriction site at the ATG start codons and a

MluI restriction site in the 3-untranslated region of the genes. This manipulation allowed cloning of the ORFs into a modified pET (pETHIS6/pET8c. Schlaich and Hurt. 1995) vector previously cut w'ith XlolI-

Mluld and created in-frame fusion proteins of six histidine residues joined

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G.Simos et al. by a spacer Ser-Ser dipeptide to the amino acid immediately after the start methionine. The vectors containing the fusion genes were transformed into Ecoli BL21 cells. Five hundred ml cultures were grown in minimal medium at 37°C to OD 0.7, shifted to 25°C and induced by the addition of I mM IPTG. The bacterial cell pellet was lysed by sonication in 10 ml of 50 mM KPi, 200 mM NaCI, 0.1% TX-l00, 10 mM P-mercaptoethanol, pH 8.0 and the lysate was cleared by centrifugation. The major part of the Pus I p fusion protein was recovered in the soluble lysate, whereas most of Loslp stayed in the pellet and resisted extraction even in the presence of 8 M urea. The protein band corresponding to Loslp was therefore purified by electroelution from SDS-polyacrylamide gels. Puslp was purified to near homogeneity by applying the soluble lysate onto a Ni-NTA resin (Qiagen, Hilden, Germany) column and elution with 150 mM imidazole in lysis buffer lacking Triton X-100. The purified proteins were dialyzed against phosphate-buffered saline and were used to immunize rabbits (100 sg per injection). Antibodies from positive sera were affinity purified using recombinant Puslp coupled to Affi-Gel 10 (Bio-Rad) or recombinant Loslp immobilized on nitrocellulose filters.

Analysis of pseudouridinylation of tRNA Recombinant Puslp to be used for enzymatic assays, after elution from the Ni-NTA column, was dialyzed against 50 mM Tris-HCI, 10 mM MgCI,, 1 mM dithiothreitol, pH 7.5, glycerol was added to 50% and stored at -80°C at a concentration of 0.6 mg/ml. The protein was diluted in the assay mixture 1/500 just prior to use. T7 transcription of tRNAs, preparation of S 100 yeast extracts, in vitro pseudouridine synthase assays and quantitation of P were performed as previously described (Szweykowska-Kulinska et al., 1994). Yeast tRNA transcripts to be used as substrates included tRNAAIa(AGC), radiolabeled with [x-32P]GTP (for testing formation of P38) or with [cz-32P]CTP (for testing formation of P55), tRNAPhe(GAA), radiolabeled with [at-32P]CTP (for testing formation of T39 and P55), tRNAASP(GUC), radiolabeled with [ct-32PIUTP or [a-32P]ATP (for testing formation of, respectively, P 13 or P32), tRNAIIe(UAU) as well as mutant tRNAIIe(UUA), both with or without an intron, radiolabeled with [a-32PIATP (for testing introndependent formation of T34, T35 and P36), and tRNAIle(UAU) without an intron, radiolabeled with [ox-32P]CTP (for testing formation of P27, P55 and P67).

Indirect immunofluorescence Immunolocalization of the ProtA-Los 1 p/Pus 1p fusion proteins and authentic proteins was done as previously described (Grandi et al., 1993). For immunofluoresence with the anti-Loslp antibody, in order to obtain a strong signal, fixation time was reduced to 20 min. Myc-Loslp was detected by the affinity-purified monoclonal 9E10 (kindly provided by Elina Ikonen, EMBL, Heidelberg, Germany), Nsplp by an affinitypurified anti-Nsplp antibody (Nehrbass et al., 1990) and nucleoporins by mAb4l4 (BAbCO, Richmond, CA).

Metabolic labeling, RNA extraction and Northern hybridization Strains s13 and s13 complemented by TFC4 were transformed with a pRS316-URA3 plasmid in order to be able to grow in minimal medium without uracil. Cells were grown to OD 0.5, and 5 ml aliquots were incubated with 50 gCi of [3H]uracil for 30 min at 30°C before harvesting the cells. Total cell extracts were prepared in the presence of cycloheximide as described in Baim et al. (1985). RNA extraction from total cell extracts, polyacrylamide gel electrophoresis and Northern hybridization were performed as described in Sharma et al. (1996).

Miscellaneous DNA manipulations (restriction analysis, end filling reactions, ligations, PCR amplifications, etc.) were done essentially according to Maniatis et al. (1982). Isolation of total yeast DNA and Southern analysis was performed essentially as described in Sherman et al. (1986). Suppressor tRNA activity was analyzed by a color colony assay as described earlier (Hopper et al., 1980) or by growth on SDC -ade plates. The SupIl, which is as tyrosine-containing and intron-carrying suppressor tRNA which suppresses ochre stop codons, was inserted into the ARS/CENand TRPI-containing plasmid pUN20 (Elledge and Davis, 1988). This plasmid also contained the NOPI-ProtA gene. Preparation of protein extracts, SDS-PAGE, Western blotting and immunoprecipitation were performed as described (Grandi et al., 1993). Nuclear protein uptake and nuclear poly(A)+ RNA export were monitored using previously described methods (Grandi et al., 1993; Nehrbass et al., 1993).

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Accession number The GenBank accession number of the PUSI DNA sequence is X80673, and of the PUS2 DNA sequence is X80674. The accession number for the LOSI DNA sequence as determined in (Shen et a!., 1993) is L13941.

Acknowledgements We are particularly indebted to Colette Simon and Daniele Foiret (GifSur Yvette, France) for their invaluable help in Pus I p enzymatic analysis. The initial help of Valerie Doye in providing the nup133- mutant is also greatly acknowledged. We are also grateful to Symeon Siniossoglou for providing the nup84- and nup.12W strains, Dr Steve Kron (Whitehead Institute, MA) for the triple myc tag, Dr Elina Ikonen (EMBL, Heidelberg, Germany) for the affinity-purified anti-Myc antibody, Dr John Walker (North Adelaide, Australia) for sending us the XEMBL3 clone with a yeast genomic insert containing the yeast carboxylase gene and the 5' adjacent gene PUS2, Drs R.Giege and F.Fasiolo (Strasbourg, France) for plasmids containing yeast tRNAASP and yeast tRNAIIe, respectively, Dr O.Uhlenbeck (Boulder, CO) for the plasmid containing tRNAPhe, C.Wimmer for providing the s1370 mutant strain, K.Helmuth for his help in raising the anti-Loslp antibodies and to the various members in the laboratory for critically reading the manuscript. E.C.H. was the recipient of a grant from the Deutsche Forschungsgemeinschaft (DFG) and H.G. was supported by grants from the Centre National de la Recherche Scientifique (CNRS) and the 'Association pour la Recherche sur le Cancer' (ARC).

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