The EMBO Journal vol.9 no.4 pp. 1 245 - 1252, 1990

Accumulation of pre-tRNA splicing '2/3' intermediates in a Saccharomyces cerevisiae mutant

Calvin K.Ho1, Reinhard Rauhut, Usha Vijayraghavan and John Abelson Divisions of Biology and 'Chemistry, California Institute of Technology, Pasadena, CA 91125, USA Communicated by G.Tocchini Valentini

In an effort to identify genes involved in the excision of tRNA introns in Saccharomyces cerevisiae, temperaturesensitive mutants were screened for intracellular accumulation of intron-containing tRNA precursors by RNA hybridization analysis. In one mutant, tRNA splicing intermediates consisting of the 5' exon covalently joined to the intron ('2/3' pre-tRNA molecules) were detected in addition to unspliced precursors. The mutant cleaves pre-tRNAPhe in vitro at the 3' exon/intron splice site, generating the 3' half molecule and 2/3 intermediate. The 5' half molecule and intron are not produced, indicating that cleavage at the 5' splice site is suppressed. This partial splicing activity co-purifies with tRNA endonuclease throughout several chromatographic steps. Surprisingly, the splicing defect does not appreciably affect cell growth at normal or elevated temperatures, but does confer a pseudo cold-sensitive phenotype of retarded growth at 15°C. The mutant falls into the complementation group SEN2 previously defined by the isolation of mutants defective for tRNA splicing in vitro [Winey,M. and Culbertson,M.R. (1988) Genetics, 118, 609-617], although its phenotypes are distinct from those of the previous sen2 isolates. The distinguishing genetic and biochemical properties of this new allele, designated sen2-3, suggests the direct participation of the SEN2 gene product in tRNA endonuclease function. Key words: Saccharomyces cerevisiae tRNA splicing mutant/tRNA processing/tRNA splicing endonuclease

Introduction Eukaryotic nuclear transfer RNAs arise from longer precursors that contain extended 5' and 3' termini and, in some specific instances, intervening sequences. Each nascent transcript is progressively trimmed and modified to the mature sized tRNA through a series of ordered nuclear processing reactions, before being transported into the cytoplasm to participate in protein synthesis (De Robertis and Olson, 1979; Melton et al., 1980). Where present, a single short intervening sequence (in most cases, 60 nucleotides or less) interrupts the precursor at a conserved position within the anticodon loop, and therefore must be spliced out in order to produce a functional tRNA (Ogden et al., 1984; Sprinzl et al., 1989). In yeast, wheat germ and Xenopus, the native substrates for tRNA splicing are fully end-trimmed and carry the 3' terminal CCA modification (Knapp et al., 1978; De Robertis and Olson, 1979; Mao Oxford University Press

et al., 1980; Stange and Beier, 1987). Intron excision is thus a relatively late step in the maturation pathway. In Saccharomyces cerevisiae, tRNA splicing is a multistep process requiring several enzymatic activities [for reviews, see Hopper (1984) and Abelson et al. (1986)]. It initiates with cleavage at the two splice sites by tRNA endonuclease, releasing the intron and generating tRNA half molecules with 2',3' cyclic phosphodiester and 5' hydroxyl termini (Peebles et al., 1983). The half molecules are covalently joined by tRNA ligase in an ATP dependent step, and the extraneous 2' phosphate produced at the ligated junction is removed by a phosphatase to generate spliced tRNA (Greer et al., 1983; McCraith and Phizicky, 1990). A single endonuclease and ligase are sufficient in S. cerevisiae to splice the nine known species of intron-containing tRNAs in vitro, and presumably in vivo as well (Peebles et al., 1983). tRNA ligase has been purified to homogeneity and comprises a single 95 400 dalton soluble polypeptide that contains the activities of 2',3' cyclic phosphodiesterase, 5' polynucleotide kinase, adenylylate synthetase and RNA ligase associated with the joining reaction (Phizicky et al., 1986). Using the N-terminal sequence of the protein, the structural gene for ligase, RLGJ, was cloned and found to encode a 827 residue basic protein (Westaway et al., 1988). The multiple activities of tRNA ligase appear to be organized linearly in distinct structural domains along the polypeptide (Q.Xu, D.Teplow, T.Lee and J.Abelson, in preparation). Comparatively less is known about tRNA endonuclease. The endonuclease activity fractionates with membranes during purification and is stimulated by non-ionic detergents (Peebles et al., 1983; Green and Abelson, 1989). Since tRNAs are spliced in the nucleus, these properties suggest that the yeast endonuclease resides at the nuclear envelope. We have recently purified the endonuclease to homogeneity, and find that it consists of three polypeptides of mol. wts 31, 42 and 51 kd (R.Rauhut and J.Abelson, in preparation). The multimeric nature of tRNA endonuclease raises interesting questions about the roles of each subunit, such as the distribution of catalytic centers for each of the two cleavage reactions, and the location of the postulated membrane interaction domain. Obviously, information on the nature and structure of the gene products of endonuclease would help clarify such issues. There have been numerous attempts to identify genes specifically involved in tRNA intron excision. The majority of these have centered on isolating conditional yeast mutants defective for tRNA splicing (reviewed in Hopper, 1989). To date, seven distinct S. cerevisiae genes-RNA], LOS], SEN], SEN2, STPI, TPDJ and PTAJ-have been implicated in the removal of introns from tRNA precursors. The methods for isolating mutants of these genes can be grouped into three approaches: (i) screening for mutants that accumulate precursor tRNAs in vivo [RNAJ, PTA] (Hopper et al., 1978; J.P.O'Connor and C.L.Peebles, personal communication)]; (ii) selecting for mutants or cloned genes 1245

C.K.Ho et al.

that alter the processing of intron-containing suppressor tRNAs, thereby rendering them phenotypically distinguishable in in vivo suppression assays [LOS], STPI, TPDJ (Hopper et al., 1980; Wang and Hopper, 1988; van Zyl et al., 1989)]; and (iii) assaying individual mutant extracts for conditionally labile tRNA splicing activities [SEN], SEN2 (Winey and Culbertson, 1988)]. While there is as yet little definitive evidence that any of these genes code for structural components of the endonuclease, some are less likely to serve this function than others, and instead probably affect the splicing machinery only indirectly. For instance, temperature-sensitive mutants of RNA], LOS] and PTA] have been found to exhibit normal tRNA splicing activities in vitro, even though these strains accumulate unspliced tRNA precursors under restrictive conditions (Winey and Culbertson, 1988; L.D.Schultz and A.K.Hopper, personal communication; J.P.O'Connor and C.L.Peebles, personal communication). Another example is the recently isolated STPI gene, which accumulates only a subset of intron carrying pre-tRNAs and is non-essential for growth (Wang and Hopper, 1988). On the other hand, mutants of SEN] and SEN2 have been shown to have thermosensitive tRNA endonuclease activity in vitro which fractionates with the endonuclease during partial purification (Winey and Culbertson, 1988). These findings suggest a more direct participation for the SEN gene products in intron excision. Our laboratory recently constructed a collection of 1000 temperature-sensitive yeast mutants with the initial aim of isolating genes involved in mRNA splicing (Vijayraghavan et al., 1989). Hybridization analysis of RNA extracted from individual mutants, using probes complementary to mRNA introns, identified a wide range of new candidates that specifically accumulated mRNA splicing precursors and intermediates. Encouraged by the success of this approach, we decided to screen members of this collection for mutants that amass tRNA precursors. In the course of screening, we discovered a mutant that accumulates pre-tRNA splicing '2/3' intermediates consisting of the 5' exon covalently joined to the intron. Since this phenotype suggested the inhibition of cleavage at one of the two splice sites in vivo, we focused our attention on characterizing this mutant in detail. Here, we report on experiments which explore the possibility that the mutant is specifically defective in the tRNA endonuclease. -

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Results Screen for pre-tRNA processing mutants In this study, 500 of the 1000 temperature-sensitive yeast mutants we recently generated by ethylmethane sulfonate mutagenesis (Vijayraghavan et al., 1989) were screened for tRNA processing mutants by RNA hybridization (Northern) analysis to detect accumulation of unspliced pre-tRNAs. The procedure consists of: (i) extracting total RNA from individual mutants grown to early to mid-logarithmic phase at permissive temperature (23°C), followed by incubation at non-permissive temperature (37°C) for 2 h before harvesting; (ii) separating the small RNAs ( 20 tetrads from a cross of sen2-3 and wild-type strains (Figure 3a and b). In addition, we examined by Northern analysis the level of tRNATYr precursors present in mid-logarithmic cells grown in rich media at 15, 23, 30 and 37°C, using oligonucleotide probes complementary to the 3' and 5' half molecules of tRNATYr.

1248

This analysis would reveal whether the retarded growth at low temperatures is paralleled by a commensurate increase in the accumulation of tRNA splicing intermediates in vivo. Indeed, intracellular levels of 2/3 and 3' half molecules were observed to increase with decreasing growth temperature (Figure 4). Concomitantly, there is also a reduction in the steady-state level of mature tRNATYr in cells grown at lower temperatures. In none of the conditions was the free 5' half molecule detected. Whole cell extracts prepared from sen2-3 and wild-type strains were assayed for tRNA splicing activity to investigate the effect of the mutation on splicing in vitro. sen2-3 extracts were found to partially splice T7 transcribed pre-tRNAPhe in the endonuclease assay, generating 2/3 and 3'-half molecule products (Figure 3c). The partial splicing activity co-segregated with the pseudo-cs and 2/3 accumulation phenotypes. No significant level of the 5' half molecule or intron was detected, indicating that the extract is unable to cleave pre-tRNA at the 5' splice site. This result is consistent with the observation that the 2/3 intermediates accumulating in vivo comprise the intron and 5' half molecules. The presence of 2 mM ATP in the assay had no appreciable effect on the partial splicing of sen2-3 extracts, and no ligated

Fig. 3. Phenotypes of sen2-3. (a) Spores (A-D) among four sets of tetrads (1 -4) generated from a SEN2'/sen2-3 diploid were germinated on YPD at 15'C to show 2:2 segregation of pseudo cold-sensitive growth. (b) Northern blot of RNAs (1 gtg) extracted from early to mid-log cultures (OD6W = 1-2) of the spores in (a) probed with a pre-tRNAL"~intron specific oligonucleotide shows the 2/3 pre-tRNA accumulation to co-segregate with pseudo cold sensitivity. (c) Cell extracts prepared from mid-log cultures (OD60o = 2-3) of spores in (a) were assayed for tRNA endonuclease activity as described in Materials and methods, using pre-tRNAPhe substrate uniformiy labeled with [ci-32p]UTP by T7 polymerase transcription. The partial in vitro splicing of precursor to the 3' half and 2/3 molecules co-segregates with the cold sensitivity in (a) and in vivo pre-tRNA accumulation in (b). The leftmost two lanes indicate the position of products generated in a mock reaction (-endo) and in the presence of purified wild-type endonuclease (+endo). Size markers (mol. wt) are from 5' 32P-labeled fragments from complete HpaII digestion of pBR322 DNA.

Yeast tRNA splicing mutant 3'-half

probe: strain

wt

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AB CD

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Fig. 4. Temperature dependence of the accumulation of tRNA splicing intermediates. Total RNA was extracted from wild-type (wt) and sen2-3 cells grown to early to mid-log (OD6W = 1-2) in YPD at 15°C (lane A), 23°C (lane B), 30°C (lane C) and 37°C (lane D). RNAs (1 Ag) were separated on a 8% polyacrylamide/8 M urea gel, blotted onto a hybridization filter, and _probed with oligonucleotides complementary to the 3' half of tRNA' Y at positions 70-94, and the 5' half of tRNATY' at positions 1-25. The uppermost three bands were identified as unspliced tRNATYr precursors in a separate experiment showing that the same RNAs are detected by a pretRNATYr intron specific probe. Mature tRNATYr was identified by hybridization of this RNA to an oligonucleotide complementary to tRNATYr across the spliced junction (positions 29-54). The other bands were assigned based on the nature of the hybridizing probe and their observed sizes relative to markers from complete HpaH digestion of pBR322 DNA.

mature tRNAPhe was produced. None the less, the tRNA ligase activity in the sen2-3 mutant is apparently unaffected, since ATP dependent ligation was observed when highly purified endonuclease devoid of ligase activity was added to an assay of the sen2-3 extract to generate 5' and 3' half molecules in situ (data not shown). Rather, the absence of ligation stems from the inability of the sen2-3 extract to generate 5' half molecules in vitro. The 2/3 pre-tRNAPbe intermediate was mapped by RNA fingerprinting to verify that the molecule terminates at the 3' splice site with the proper 2',3' cyclic phosphodiester end produced by endonuclease. Endonuclease partially purified from the sen2-3 mutant (see below) was used to generate ATP-labeled 2/3 pre-tRNAph' from precursor transcribed with [c-32P]ATP. The 2/3 molecule was gel-purified and digested with RNase TI. The TI fragments were then separated by electrophoresis on cellulose acetate membrane followed by homochromatography on polyethyleneimine (PEI) thin layer plates. Upon correct cleavage at the 3' splice site by endonuclease, the 3' terminal U of 2/3 pre-tRNAphe becomes labeled by nearest neighbor phosphate transfer from the 5' terminal A of the 3' exon. Nearly stoichiometric amounts of the 3' terminal UU > p fragment were observed in the fingerprint ( - 0. 8 mol per mol 2/3 molecule), indicating correct termination at the 3' splice site (Figure 5a). The presence of the terminal 2',3'-cyclic phosphate was verified by secondary analysis of UU > p fragment eluted from the plate. Digestion with nuclease P1 followed by calf intestinal phosphatase quantitatively liberated labeled uridine

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Fig. 5. RNA sequence analysis of the pre-tRNAPhe 2/3 intermediate. (a) Gel isolated [a-32P]ATP labeled 2/3 pre-tRNAPhe was digested to on completion with RNase Ti and fingerprinted as described in Materials and methods. The orientation of each dimension (1, electrophoresis shown. cellulose acetate pH 3.5; 2, homochromatography on PEI-cellulose thin layer plate) and the position of xylene cyanol marker (XC) are as Minor fragments tl and t2 are UU2TP, and a mixture of UU2'P + UU3'P respectively, that arise from spontaneous opening of the cyclic phosphate of UU > p during the chromatography, based on the migration previously observed for RNA fragments with acyclic versus cyclic ends (Peebles et al., 1983). (b) Sequence of the 2/3 segment (5' exon + intron) of pre-tRNAPhe. The arrows mark the splice sites. Ti cleavages are denoted by slashes in UU>p (/) and fragments labeled by transcription of the precursor with [a-32P]ATP are underlined. (c) The structure of the terminal phosphate fragment eluted from the PEI plate in (a) was analyzed by treatment with nuclease P1 followed by calf intestinal phosphatase (CIP). In the scheme (P,) marker (lane 2) on the left, p denotes radiolabeled phosphate. The products of the secondary analysis (lane 1) and [32P]inorganic phosphate uridine 2',3' cyclic were chromatographed on a PEI-cellulose plate developed with 0.5 M LiCl. The position of the origin (ori) and unlabeled monophosphate (U > p) marker are indicated to the right of the autoradiograph. The top of the autoradiograph corresponds to the solvent front.

1249

C.K.Ho et al.

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Fig. 6. In vitro tRNA splicing activity of endonucleases partially purified from sen2-3 and wild-type (wt) yeast. The peak activity fraction from heparin-agarose chromatography was assayed for splicing of [a-32P]UTP labeled pre-tRNAPhe as described in Materials and methods. Reaction aliquots were taken at the times indicated and splicing products were resolved by electrophoresis on a 10% acrylamide/4 M urea gel. In the last lane, equal volumes of peak fractions from heparin-agarose chromatography of sen2-3 and wildtype enzymes were mixed, followed by a 15 min assay of endonuclease activity.

2',3' cyclic monophosphate (Figure Sc). Only a small fraction of the label was present in free phosphate, indicating that few of the UU molecules terminate in a phosphatasesensitive acyclic phosphomonoester.

Co-purification of mutant splicing with tRNA endonuclease To determine whether the 2/3 splicing activity is directly due to an altered tRNA endonuclease rather than to some other extrinsic factor in the mutant, the endonuclease was extensively purified from sen2-3 yeast and its splicing activity was examined. The activity extracted from membranes and eluted from the heparin -agarose column was found to cleave pre-tRNAPhe at the 3' splice site only, generating 2/3 and 3' half molecules (Figure 6). The 5' half and intron were not produced, even after 2 h incubation. 2/3 splicing activity was found to have chromatographic properties (e.g. binding capacity, elution profile) identical to wild-type tRNA endonuclease in this and each of the following chromatographic steps. It is estimated that upon final fractionation on phospho-Ultrogel, endonuclease has been purified 8000-fold relative to the activity in crude membranes. The possibility that 2/3 splicing results from a co-purifying inhibitor was ruled out by showing that mixed fractions of purified sen2-3 and wild-type endonuclease generate normal reaction products (Figure 6). These results collectively argue that the 2/3 splicing arises directly through the action of an altered tRNA endonuclease in sen2-3 cells.

Discussion We have established the following phenotypes for sen2-3: (i) accumulation of pre-tRNA '2/3' splicing intermediates

1250

in vivo for at least five of the nine intron-carrying species of tRNA; (ii) partial cleavage of pre-tRNAPhe to generate 2/3 and 3' half molecules in vitro; and (iii) slow growth at low temperatures. The splicing intermediates are composed of 5' exon and intron segments, and thus reflect the absence of cutting at the 5' splice site. Although it is not known whether tRNA endonuclease cleaves pre-tRNA in a specific order, the nature of the 2/3 splicing activity implies that cleavage at the 5' splice site is not an obligatory leading step in intron excision. This view is also supported by the observation that a low amount of joined 5' exon/intron molecules appears during splicing by wild-type endonuclease (Peebles et al., 1983; Reyes and Abelson, 1988). In addition, several point mutations in the mature domain of pretRNAPhe have been shown to block 5' cleavage, but still permit cleavage at the 3' splice site (Reyes and Abelson, 1988). Although it can be argued that the 2/3 species accumulates as a dead end product, we have found that pretRNAphe singly cut in vitro at the 3' splice site by the sen2-3 endonuclease can be further processed to half-molecules by the wild-type enzyme (unpublished data). This suggests that the 2/3 molecule, when properly annealed to the 3' exon, is a true biochemical intermediate of the splicing pathway. The RNAs accumulating in sen2-3 cells are exclusively intron containing tRNAs, indicating that the defect lies specifically in the tRNA splicing pathway. Because splicing is essential, it is somewhat surprising that sen2-3 mutants are viable under a number of conditions, and are only mildly growth inhibited at 15°C. The viability can be explained by supposing that sen2-3 does not completely block 5' splice site cleavage, so that the mutant is able to generate enough spliced tRNA to support growth. RNA hybridization analysis confirms that a significant amount of mature tRNATYr is present among the small RNAs in actively growing sen2-3 cells, albeit at a level lower than in wild-type yeast. Additional support comes from the observation that sen2-3 does not affect the amber or ochre suppression efficiency of tRNAUCG when suppressor alleles of this gene are expressed in sen2-3 cells from centromere plasmids (Ho and Abelson, 1988), indicating that the intron-carrying precursors to this tRNA are properly spliced in vivo (unpublished data). In light of the cold-sensitive growth of sen2-3 strains, it is of interest to note the reports of cold-sensitive mutants in ribosomal RNA processing. Mutants of Escherichia coli that accumulate pre-ribosomal particles in vivo are sometimes associated with cold sensitivity (reviewed in Jaskunas et al., 1974). In yeast, strains lacking snRIO, a small nuclear RNA participating in rRNA processing, grow poorly and accumulate rRNA precursors at low temperature (Tollervey and Guthrie, 1985; Tollervey, 1987). In both instances, coldsensitive growth is thought to arise from inhibition of ribosome assembly, a process known to require a substantial heat of activation in vitro (Jaskunas et al., 1974). It is unlikely, however, that the cold sensitivity of sen2-3 stems from an analogous defect in assembling the subunits of tRNA endonuclease and/or ligase into a functional splicing complex, since the chromatographic properties of the sen2-3 endonuclease are indistinguishable from that of the wild-type enzyme, and ligase activity is unaffected in the mutant. Rather, the growth limiting factor is more likely to involve a temperature-dependent effect on the catalytic efficiency of the sen2-3 endonuclease that promotes the greater accumulation of tRNA splicing intermediates, and a correspondingly

Yeast tRNA splicing mutant

reduced level of mature tRNAs, found in cells grown at low temperatures. Considering the lipophilic properties of tRNA endonuclease, known temperature effects on the lipid composition of yeast membranes (reviewed in Henry, 1982) could play a role in amplifying the aberrant splicing activity. Co-purification of 2/3 splicing with the endonuclease provides compelling evidence for a structurally altered enzyme in the mutant, and rules out alternatives in which sen2-3 affects splicing indirectly by altering a soluble ancillary activity in the tRNA splicing pathway. An example would be the inactivation of a degradative ribonuclease that scavenges tRNA splicing intermediates prematurely released from the endonuclease. Although the co-fractionation result points towards sen2-3 encoding a mutant subunit of tRNA endonuclease, we cannot rule out the possibility that the mutation inactivates an enzyme required for proper posttranslational processing of the endonuclease (e.g. glycosylation or proteolytic processing). However, any such processing defect would have the rather remarkable property of selectively inhibiting one of the cleavages while leaving the other unaffected. sen2-3 falls into one of two complementation groups, SEN] and SEN2, previously defined through the isolation of mutants defective for tRNA splicing in vitro (Winey and Culbertson, 1988). Mutants carrying senl-l are deficient in tRNA endonuclease activity in small scale cell extracts under a variety of conditions tested, accumulate intron-containing pre-tRNAs in vivo and fail to grow at 37°C. Although yielding thermolabile splicing activity in vitro, mutants carrying sen2-1 in contrast do not accumulate pre-tRNAs, nor is there any temperature effect on growth. Based on the less pronounced phenotypes of sen2-1, Winey and Culbertson (1988) have suggested that SEN2 could specify an accessory function that activates or enhances the core endonuclease activity encoded possibly by SEN]. However, it was noted that without knowing the phenotype of a SEN2 deletion mutant, one cannot rule out that catalytic activity is specified by SEN2. The phenotype of selective inhibition of 5' splice site cleavage in sen2-3 and the co-purification of this activity with tRNA endonuclease point toward the latter possibility, in which the SEN2 gene product carries out at least one of the cleavages. Further evidence for an integral role in endonuclease function comes from our recent cloning of SEN2 by complementation of the cold-sensitive phenotype. The gene was found to be essential for growth and to encode a putative polypeptide close in size to the 42 kd subunit of endonuclease (C.K.Ho, R.Rauhut and J.Abelson, in preparation). Should the SEN2 gene product prove to be this subunit, the yeast mutant reported here, together with the complementing gene, will provide new opportunities for understanding the structure of tRNA endonuclease, its mode of action and its location in the cell.

Materials and methods Strains and plasmids Table I lists the yeast strains used in this study. Plasmids YCpRNA1, YEpRNAl, YCpLOS I and YEpLOSl (Atkinson et al., 1985; Hurt et al., 1986) were obtained from S.Wang and A.Hopper (Pennsylvania State University). pEP9 and pEPI 1, containing the yeast RLG+ (tRNA ligase) gene on episomal and centromere plasmids respectively (Westaway et al., 1988), were obtained from S.Westaway (California Institute of Technology). Yeast transformation was performed using the lithium acetate method (Ito et al., 1983).

Media, growth and genetic analyses Rich (YPD) and supplemented minimal media were prepared according to Sherman et al. (1986). Sensitivity to high temperature was tested by replica plating cell patches onto YPD plates and incubating at 37°C for up to 3 days. Cold sensitivity was tested by streaking cells on YPD plates and incubating at 15°C for up to 5 days. Growth rates were measured for cells grown in liquid media using Klett flasks (Bellco) and Klett meter (KlettSummerson). Osmotic sensitivity was tested by streaking cells onto YPD plates supplemented with 0.5-1.5 M KCI, 0.75 M NaCl or 2.5 M glycerol, and growth at low pH was tested on YPD plates adjusted to pH 3.5-5.5 with 1 N HCI or 0.2 M acetic acid, as described by McCusker et al. (1987). Yeast haploid mating and selection of diploids by complementation of auxotrophic mutations were performed at 30°C as described by Vijayraghavan et al. (1989). Diploids were sporulated at 30°C in liquid presporulation and sporulation medias (Sherman et al., 1986), and spores were dissected on YPD plates using standard micromanipulation procedures.

Temperature-sensitive bank and RNA isolation - 1000 independent temperature-sensitive S. cerevisiae mutants using ethyl methanesulfonate mutagenesis is described by Vijayraghavan et al. (1989). In this study, - 500 members of the bank derived from mutagenizing strain SS330 were analyzed for accumulation of unspliced precursor tRNAs in vivo. Cultures (2 ml) of individual mutants Construction of

were grown in rich media to

early

to

mid-logarithmic phase (OD6W

=

0.8-1) at 23°C (permissive condition), and shifted to and incubated at 37°C (non-permissive condition) for 2 h. Total RNA was isolated from the harvested cells by hot phenol extraction as described by Vijayraghavan et al. (1989). The RNA concentration was quantitated by measuring OD260. RNA hybridization (Northern) analysis Total RNA (1 Ag) was denatured by heating at 100'C for 2 min in 95% formamide, 20 mM Tris-HCI pH 8.0, 5 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol, and resolved by electrophoresis in 8% 29:1 acrylamide:bis-acrylamide/8 M urea gels in 89 mM Tris-borate pH 7.5, 20 mM EDTA buffer at 25 mA. RNAs were transferred onto a GeneScreen hybridization filter (NEN Research Products) by electroblotting using a Biorad apparatus, and fixed by UV crosslinking, as described by Cheng and Abelson (1987). Filter prehybridization and hybridization with 5' 32P-labeled oligonucleotide probes were carried out according to Newman et al. (1983). The filters were washed three times for 5 min each at 25°C in 6 x SSC buffer (0.9 M sodium chloride, 0.09 M sodium citrate pH 7.0), and autoradiographed overnight at -70°C with intensifying screen. tRNA splicing extracts Small scale whole cell extracts were prepared from 40 ml mid-logarithmic cells (OD6W = 2-3) grown in YPD at 30°C, as described by Phizicky et al. (1986), except that the spheroplasts were homogenized in 50 ju buffer containing 20 mM potassium phosphate pH 7.5, 0.2 M NaCl, 2 mM EDTA, 10 mM DTT, 1 mM PMSF, 10% glycerol and 0.5% Triton X-100 (Sigma), and the final centrifugation was omitted. To assay for tRNA endonuclease activity, 1.5 1l of extract was incubated for 10 min at 30°C in 8.5 jil of reaction cocktail containing 20 mM sodium HEPES pH 7.5, 5 mM MgCI2, 2.5 mM spermidine-HCI pH 7.5, 0.1 mM DTT, 0.4% Triton X-100 and 2000-5000 c.p.m. synthetic pre-tRNAPhe uniformly labeled with [a-32P]UTP (40 uCi) by T7 polymerase transcription according to Reyes and Abelson (1987). tRNA ligase activity was assayed by including 2 mM ATP in the splicing cocktail. The reaction was terminated by adding 1 I1 2% SDS, 100 mM EDTA and 2 mg/ml proteinase K, and incubating at 50°C for 10 min. The RNA was extracted with an equal volume of phenol equilibrated with 50 mM sodium acetate pH 5.0, 10 mM EDTA, and precipitated with 3 vol ethanol. The RNA was resuspended in S 1I of 1 mM Tris-HCI pH 8.0, 0.1 mM EDTA, 4 M urea, 10% sucrose, 0.025% bromophenol blue and 0.05% xylene cyanol, heated at 65°C for 5 min, and separated by electrophoresis in 10% 29:1 acrylamide:bis-acrylamide/4 M urea gels. Products of the tRNA splicing reaction were visualized by

autoradiographing overnight

at

-70°C

with

intensifying

screen.

Purification of tRNA endonuclease sen2-3 yeast (strain 373-3c) was grown in 360 1 of YPD media at 30'C to OD6W = 3.5. The harvested cells were stored at -70°C. Total yeast membranes were prepared from the cells according to Wiederrecht et al. (1987). All subsequent procedures were performed at 4'C. 160 g of membranes were washed by consecutive homogenization (using a Potter-Elvehjem homogenizer) and centrifugation (42 000 r.p.m. x 2 h, Ti45 rotor), once in 100 mM Tris-HC1 pH 8.0, 20 mM EDTA, 5 mM spennidine, 1.5 mM DTT, 1 M ammonium sulfate, 10% glycerol, 1.5 mM PMSF (buffer A), and twice in 25 mM Tris-HCI pH 8.0, 1 mM EDTA,

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C.K.Ho et al. 5 mM 2-mercaptoethanol, 20% glycerol, 0.02% Triton X-100 (BoehringerMannheim), 0.2 mM PMSF (buffer B). Membranes were then extracted in buffer B containing 0.9% Triton X-100 and centrifuged as above. The supernatant was applied batchwise to heparin-agarose, material binding to the resin was washed with 25 mM Tris-HCl pH 8.0, 1 mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol, 0.9% Triton X-100, 0.2 mM PMSF (buffer C) + 0.1 M NaCl, and the resin was poured into a 2.5 x 19 cm column equilibrated with the same buffer. A 2 x 210 ml gradient of 0.1-0.7 mM NaCl in buffer C was applied to the column at a rate of 60ml/h. Active fractions eluting at 0.45 M NaCl were combined, diluted 4 x with 25 mM potassium phosphate pH 7.75, 1 mM EDTA, 5 mM DTT, 5 mM 2-mercaptoethanol,10% glycerol, 0.9% Triton X-100 (buffer D) and applied to a 2.5 x 19 cm hydroxylapatite-HA Ultrogel (IBF Biotechnics) column equilibrated with buffer D. Bound material was eluted with a 2 x 250ml gradient of 25-400 mM potassium phosphate in buffer D at a rate of 40ml/h. Endonuclease activity eluted at 200 mM potassium phosphate. Active fractions were dialyzed against 50 mM potassium acetate pH 5.5, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, 10mM MgCl2, 10% glycerol, 0.25% Triton X-100, 0.2 mM PMSF (buffer E) and applied to a 1.2 x 8.6 cm hydrazinyl tRNA-Sepharose column equilibrated with buffer E. The resin was prepared by coupling total yeast tRNA (Boehringer-Mannheim) to cyanogen bromide activated Sepharose 4B (Pharmacia) according to Remy et al. (1972). A 2 x 25ml gradient of 0-0.8 M ammonium sulfate in buffer E was applied to the column at a rate of 12ml/h. Active fractions eluting at 0.3 M ammonium sulfate were combined, diluted 3 x in buffer E and applied to a 1.2 x 8.4 cm 5' AMP-Sepharose (Pharmacia) column equilibrated with buffer E. The column was eluted with a 2 x 25ml gradient of 0-0.8 M ammonium sulfate in buffer E at a rate of 12ml/h. Active fractions eluting at 125 mM ammonium sulfate were diluted 3 x in buffer E, applied to a 1.2 x 8.4 cm phospho-Ultrogel A6R (IBF Biotechnics) column equilibrated in buffer E and eluted with a 2 x 25 ml gradient of 0-0.5 M ammonium sulfate in buffer E at a rate of 15 in/h. Endonuclease eluting at 75 mM ammonium sulfate was stored in the elution buffer at -70°C. Under these conditions, activity was retained for at least 6 months. In the final preparation the tRNA endonuclease is purified - 8000-fold relative to the activity in crude membranes with a yield of 3%. Purification of wild-type endonuclease from strain EJIOl was carried out using the same procedures. The chromatographic behavior of the wild-type enzyme was identical to that of endonuclease from the sen2-3 mutant in all column steps.

2/3 pre-tRNA sequence analysis ATP labeled pre-tRNAPhe 2/3 intermediate was prepared by treating pretRNAPhe transcribed using T7 polymerase and [a-32P]ATP (40 sCi) with sen2-3 endonuclease eluted from the heparin-agarose column. The 2/3 molecule was resolved on a 10% acrylamide (29:1)/4 M urea gel, eluted, and digested with 10 U RNase TI (Boehringer-Mannheim) in the presence of 20 Asg unlabeled yeast RNA carrier (BDH Chemicals) for 1.5 hat 37°C. Tl fragments were chromatographed by two-dimensional RNA fingerprinting according to Volckaert et al. (1976). RNA fragments were resolved in the first dimension by electrophoresis on cellulose acetate membrane (Schleicher and Schuell) pH 3.5. Separation in the second dimension is by homochromatography on PEI-cellulose thin layer plates (Macherey-Nagel). Fragments were quantitated by cutting out the spots and measuring the Cerenkov radiation. For secondary analysis, fragments were eluted from PEI plates with 30% triethylammonium carbonate pH 10 according to Volckaert et al. (1976), digested with 1 jig nuclease P1 (BoehringerMannheim) followed by 1 U calf intestinal phosphatase (BoehringerMannheim), and separated by TLC on PEI/UV254-cellulose plates (Macherey-Nagel) developed with 0.5 M LiCl. Uridine 2',3' cyclic monophosphate marker was obtained from Sigma.

Acknowledgements We thank S.Westaway, S.Wang and A.Hopper for providing plasmids; M.Winey, J.Hendrick and M.Culbertson for supplying yeast strains and for identifying spores carrying sen2-1 in the SEN2 allelism analysis; and P.O'Connor, C.Peebles, W.H.van Zyl, J.Broach, S.Wang, A.Hopper, M.Winey, J.Hendrick, M.Culbertson, S.McCraith and E.Phizicky for helpful discussions and/or communicating unpublished work. We also thank E.Phizicky for advice on preparing tRNA splicing extracts, and T.H.Chang for comments on the manuscript. C.K.H. was a National Science Foundation predoctoral fellow. R.R. was supported by a Procter and Gamble postdoctoral fellowship. This research was supported by grants from the National Institutes of Health (GM 32637) and the American Cancer Society (MV 318F).

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November 24, 1989