TheKIN28Gene is Required both for RNA Polymerase II Mediated Transcription and Phosphorylation of the Rpb1p CTD

JMB—MS 566 Cust. Ref. No. YAN 007/95

[SGML] J. Mol. Biol. (1995) 249, 535–544

The KIN28 Gene is Required both for RNA Polymerase II Mediated Transcription and Phosphorylation of the Rpb1p CTD Jean-Gabriel Valay1, Michel Simon1, Marie-Franc¸oise Dubois2 Olivier Bensaude2, Ce´line Facca1 and Ge´rard Faye1* Institut Curie-Biologie, Bax t 110, Centre Universitaire 91405 ORSAY CEDEX France

1

2

Laboratoire de Ge´ne´tique Mole´culaire, Ecole Normale Supe´rieure, 46 rue d’Ulm 75230 PARIS CEDEX 05 France

*Corresponding author

Kin28p, associated with cyclin Ccl1p, is a putative cyclin-dependent kinase (CDK) of the p34cdc2 family in Saccharomyces cerevisiae. Search for mutations co-lethal (syn mutations) with a kin28 thermosensitive mutation (kin28-ts3) has uncovered genetic interactions between gene KIN28 and genes RAD3, SIN4, STI1 and CDC37. The genetic interaction between KIN28 and the CDC37 cell division cycle gene suggests that a connection exists between the activity of CDK-Kin28p and cell-cycle progression. Both RAD3 and SIN4 gene products are implicated in the RNA polymerase II transcription process. Here we show that RNA polymerase II transcription is drastically reduced in a kin28-ts mutant, at restrictive temperature. This impairment correlates with a markedly decreased phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Rpb1p). Thus, the Kin28 gene product is required in vivo for RNA polymerase II phosphorylation and transcriptional activity as recently suggested by experiments using an in vitro reconstituted system. Keywords: C-terminal domain; RAD3; SIN4; STI1; CDC37

Introduction The Kin28 protein (Kin28p), a putative serinethreonine protein-kinase of Saccharomyces cerevisiae (Simon et al., 1986), shares 38% and 47% homology with Cdc28p (Lo¨rincz & Reed, 1984) and Mo15p of Xenopus (Shuttleworth et al., 1990), respectively. Cdc28p, associated with cyclins, controls all major cell cycle events of S. cerevisiae (Nasmyth, 1993). Mo15p, a CDK (cyclin-dependent kinase) activating kinase (CAK), activates the p34cdc2-like kinases (Poon et al., 1993; Fesquet et al., 1993). Since Kin28p interacts directly with the Ccl1p cyclin, we have postulated that Kin28p is a CDK (Valay et al., 1993). Despite the fact that the members of this kinase group characterized to date are involved in cell cycle regulation, thermosensitive kin28 mutants (kin28-ts) do not show a characteristic phenotype of cell-divAbbreviations used: CDK, cyclin-dependent kinase; CTD, C-terminal domain; Rpb1p, the largest subunit of RNA polymerase II; CAK, CDK activating kinase; TFIIH, general transcription factor IIH; NER, nucleotide excision repair; syn, synthetic lethal mutation; form IIa , Rpb1p unphosphorylated form; form IIo , Rpb1p multi-phosphorylated form; Hsp, heat shock protein; PCR, polymerase chain reaction. 0022–2836/95/230535–10 $08.00/0

ision-cycle (cdc) mutants, i.e. a complete block at a unique point of the cell-cycle (Pringle & Hartwell, 1981). On the contrary, the activity of Kin28p seems necessary at different points of the cell cycle, which is rather compatible with a metabolic defect (Valay, 1994; Valay et al., unpublished results). In the present work, we have attempted to identify processes in which Kin28p is involved. Through a search and characterization of mutations co-lethal with a kin28-ts mutation, we have identified six genes interacting with KIN28. Two of them encoded the general transcription factors RAD3 and SIN4 (Guzder et al., 1994; Jiang & Stillman, 1992). Furthermore, we have found that the transcription of eight genes, chosen at random, is drastically affected upon shift to restrictive temperature in a strain carrying a kin28-ts mutation. In the transcription model generally accepted, the Rpb1p subunit of RNA polymerase II cycles from an unphosphorylated state taking part in transcription initiation to a highly phosphorylated state acting in RNA chain elongation (Drapkin & Reinberg, 1994; Dahmus, 1994). The genetic interaction between KIN28 and RAD3 suggested that Kin28p might be a kinase associated with the TFIIH general transcription factor (Feaver et al., 1991), which phosphorylates the 7 1995 Academic Press Limited

JMB—MS 566 536 C-terminal domain (CTD) of subunit 1 of RNA polymerase II (Rpb1p). We have found that the ratio between the CTD highly phosphorylated state and its unphosphorylated one is markedly reduced in a strain carrying a kin28-ts mutation upon shift to restrictive temperature. Taken together, our in vivo results strongly suggest that Kin28p is a CTD kinase or an activator of CTD kinases. Indeed, while this work was in progress, Feaver et al. (1994) identified Kin28p as being the kinase associated with the purified general transcription factor TFIIH.

Results Isolation of synthetic lethal mutations (syn) A priori, Kin28p could phosphorylate one or several substrates, it could be included in a multicomponent complex to bring about its function and/or be subject to activating or inhibiting factors. Therefore, we anticipated that the alteration of one or several genes, besides the kin28-ts3 mutation (J.-G.V. et al., unpublished results), would produce a synergic effect leading to lethality. The identification of such a component, interacting with Kin28p and whose function had been already elucidated, would give clues about the function(s) of Kin28p. We used a colony-sectoring assay (Bender & Pringle, 1991) to isolate mutations which are lethal in the presence of the kin28-ts3 mutation at permissive temperature. The leu2 ade2 ade3 kin28-ts3 yeast cells are white, but turn red when transformed with vector pJG72 carrying a selective marker (LEU2), the screening marker ADE3 and a copy of the KIN28 wild-type allele. This plasmid, being not essential in such cells at permissive temperature (26°C), is easily lost, generating white sectors in otherwise red colonies of plasmid-containing cells. Therefore, uniformly red colonies, i.e. those issued from cells that cannot lose pJG72, are assumed to contain mutations which are co-lethal with the kin28-ts3 mutation. Screening of 76,000 UV-mutagenized clones gave rise to 40 and 43 red mutants from strains GF314-26C (pJG72) and GF314-17B (pJG72), respectively. These mutants were still able to produce sectored colonies when transformed with plasmid pJG38, which carries the URA3 selective marker and a copy of the KIN28 wild-type allele. Complementation analysis was carried out by crossing the mutants issued from GF314-26C (pJG72) with those of GF314-17B (pJG72). Fifty-three mutants appeared to belong to nine complementation groups, each containing 2 to 17 mutants. Thirty mutants were left unassigned. Seventeen mutants were allelic to KIN28, 14 to CCL1 (Valay et al., 1993) and two to MCA28, the gene of a putative serine-threonine protein-kinase able, when borne on a multicopy vector to suppress kin28-ts mutations (Valay, 1994; J.-G.V. et al., unpublished results). None of the synthetic lethal mutations (syn) was complemented by vector YEp24 containing CDC28 (Lo¨rincz & Reed, 1984).

Cyclin-dependent Kinase Kin28p and Transcription

Thermosensitive and UV-sensitive syn mutants The use of a positive screen renders easier the identification of the syn genes. Thus, we searched for those which were thermosensitive or UV-sensitive. Six syn mutants appeared to be thermosensitive, one being allelic to MCA28. Each syn-ts mutant was mated with either strain GF314-26C or GF314-17B and tetrad analysis was performed (at least ten tetrads were scored for each syn-ts mutant) to determine whether thermosensitivity and synthetic lethality could be dissociated. For syn184, 627, 732 and 858 thermosensitivity did not segregate from synthetic lethality, whereas two mutations seem necessary to produce both phenotypes of syn151. Wild-type alleles of four of these syn-ts alleles were cloned by complementation with a wild-type centromeric library. Thermoresistant transformants were selected for. They were able to form sectored colonies at permissive temperature. From syn151 we isolated two complementing plasmids, p151-1 and p151-7, with overlapping inserts. syn559, allelic to syn151, also formed sectored colonies when transformed with these two plasmids. A one-step disruption analysis, carried out with a Mini-Mu derivative (Materials and Methods), revealed that the gene complementing syn151 and syn559 is STI1 (Nicolet & Craig, 1989). A gene homologous to STI1 exists in human and chicken cells (Honore´ et al., 1992; Smith et al., 1993). STI1 appears to be an important component of the system(s) allowing growth of yeast cells under stressful conditions. Thermoresistant transformants obtained with syn858 permitted the isolation of a plasmid, p858-17, which gave rise to sectored colonies in transformed 172, 145, 197 and 662 syn mutants, which are allelic to syn858. The complementing gene, identified by Mini-Mu disruption, is SIN4 = TSF3 = SDI3 (Jiang & Stillman, 1992; Chen et al., 1993; Stillman et al., 1994). SIN4 encodes a 111 kDa protein that is a global transcriptional regulator required for the activation of some genes and the repression of others. Strains with a sin4 mutation display a number of pleiotropic phenotypes common to histone and spt mutations (SPT, for suppressor of Ty:Winston & Carlson, 1992), suggesting that the sin4 mutation alters chromatin structure, which in turn leads to transcriptional defects. Thermoresistant transformants obtained with syn184, allowed the isolation of plasmids p184-11 and p184-18 containing overlapping inserts. The complementing gene, identified by Mini-Mu disruption is CDC37 (Reed, 1980). Temperature-sensitive mutations in this gene arrest cell cycle at ‘‘Start’’ in G1. syn184 is the unique representative of its complementation group. We have searched for UV sensitivity among the syn mutants isolated. We found five of them to be clearly UV-sensitive, particularly syn581 (Figure 1). To identify the gene affected, syn581 was mated with strains harbouring a rad mutation. The following crosses demonstrated that syn581 is allelic to rad3 (Naumovski & Friedberg, 1982). Strain FF18-358,

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Cyclin-dependent Kinase Kin28p and Transcription

gave rise to sectored colonies. We have gathered the identified complementation groups in Table 1. Kin28p and RNA polymerase II transcription

Figure 1. Radiation sensitivity of strains JGV4 (w), GF262-2 (Q) and SYN581 (R). Cells from stationary phase cultures were diluted to appropriate concentrations, plated on solid YPD and UV-irradiated. They were left five days at 26°C before observation.

carrying the rad3-e5 mutation was crossed with GF314-17B and syn581 was crossed with GF314-26C. The diploids obtained were UV-resistant, showing that the syn581 and Rad3-e5 mutations are recessive, whereas the diploid obtained by crossing syn581 with strain FF18-358 was as UV-sensitive as both haploid parents. Diploids syn581 × GF314-26C and syn581 × FF18-358 were forced to sporulate. Tetrad analysis of segregants from the former diploid showed that UV sensitivity and synthetic lethality segregate as a unique marker. All the segregants obtained from the latter diploid were UV-sensitive, confirming that syn581 and rad3-e5 are allelic. Mutants syn48, 93 and 504, which are allelic to syn581 were transformed with plasmid p522 carrying a wild-type copy of RAD3. As expected, tranformants

Table 1. Identified complementation groups Gene

syn alleles

CCL1

42, 131, 171, 188, 204, 557, 694, 784, 793, 857, 884, 890, 910, 959 71, 782 (ts) 151 (ts), 559 48 (UV), 93 (UV), 504, 581 (UV) 172, 195, 197, 662, 858 (ts) 184 (ts)

MCA28 STI1 RAD3 SIN4 CDC37

(ts), Thermosensitive. (UV), UV-sensitive. Mutants designated by a number below 500, were isolated from strain GF314-26C (pJG72).

The fact that syn858 (SIN4) and syn581 (RAD3) are co-lethal with kin28-ts3 suggested that the function of KIN28 might be related to the transcription process. Guzder et al. (1994) have shown that in a rad3 mutant (rad3-ts14) poly(A)+RNA synthesis is drastically inhibited at restrictive temperature. Since Kin28p genetically interacts with Rad3p, we have tested whether Kin28p is necessary for RNA polymerase II transcription. Strains kin28-ts3 and GF262-2 were grown at 38°C for various periods of time. Total cellular RNA was extracted and the steady-state level of mRNAs encoded by five of the genes previously studied by Guzder et al. (1994): RAD23, TRP3, ACT1, CDC9 and MET19, to which we added RP51A (Teem & Rosbash, 1983), PROT1 and H2A (White et al., 1986), was followed by Northern blot analysis (Figure 2). The steady-state level of a given mRNA is the result of its synthesis and its degradation. When synthesis is blocked, the mRNA level decreases at a speed related to its half-life (Herrick et al., 1990). Assuming that the half-life of the different mRNAs studied is not modified in the kin28-ts3 strain, the following conclusions can be drawn. First, the steady-state level of the eight genes studied, i.e. most probably their synthesis, diminishes as the incubation time at 38°C increases. Second, the transcription of the various mRNA probed is affected differently in the kin28-ts3 mutant compared to mutant rad3-ts14 (Guzder et al., 1994). Whereas the inhibition of ACT1 gene transcription is similar in both mutants, the steady-state levels of CDC9 and TRP3 mRNAs are less depressed in mutant kin28-ts3 than in rad3-14. In the former mutant, CDC9 mRNA seems to reach its minimum (315%) after 30 minutes at 38°C as TRP3 mRNA does (20%) after 45 minutes at 38°C. Kin28p and the phosphorylation of the CTD of Rpb1p Several subunits of the transcription complex are phosphorylated (Bre´ant et al., 1983; Kolodziej et al., 1990), particularly the largest subunit of RNA polymerase II (Rpb1p), whose carboxy-terminal domain (CTD) is composed of a 26-fold heptapeptide repeat (Allison et al., 1985). Five of the seven amino acid residues are putative phosphorylation sites. The Rpb1p unphosphorylated form (form IIa ) migrates faster than the multi-phosphorylated form (form IIo ) in polyacrylamide gels. Both forms IIa and IIo of Rpb1p could be detected in Western blots by using a monoclonal antibody raised against the CTD. The Kin28p has been found to be a CTD kinase in vitro (Feaver et al., 1994). Therefore, we have tested the involvement of Kin28p in CTD phosphorylation in vivo. In wild-type cells grown at 24°C or shifted to 39°C and in mutant kin28-ts3 cells maintained at

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Figure 2. Effect of mutation kin28-ts3 on mRNA levels. The messenger RNA level of genes RAD23, CDC9, MET19, TRP3, ACT1, RP51A, PROT1 and H2A in kin28-ts3 and KIN28 strains grown at 38°C, was followed at the indicated times by Northern hybridization.

permissive temperature, the forms IIa and IIo are present in comparable amounts (Figure 3). In contrast, the intensity of the IIo form band falls rapidly ten to 20 minutes after transfer of the mutant cells to 39°C (Figure 3, kin28-ts3, 10'). Provided the activity of the phosphatase(s), which dephosphorylates the CTD, is not affected in the kin28-ts3 strain, this result indicates that the CTD phosphorylation is blocked by the kin28-ts3 mutation at restrictive temperature.

Discussion We have attempted to gain insights into the function(s) of Kin28p by seeking mutants (syn mutants) which are not viable at 26°C when the mutation they harbour is associated with the conditional kin28-ts3 mutation (synthetic lethality), then by cloning the wild-type genes complementing the synthetic lethal mutants. This approach uncovered interactions between KIN28 and genes whose products should interact physically or functionally.

Figure 3. Phosphorylation state of RNA polymerase II subunit 1 (Rpb1p) in crude extracts prepared from strains KIN28 and kin28-ts3. Cells were grown 0, 10, 20, or 30 minutes at 39°C. Similar amounts of total protein were electrophoresed on a 5% polyacrylamide/SDS gel and detected by Western blot using a monoclonal antibody directed against the CTD. The unphosphorylated form (IIa ) migrates faster than the multi-phosphorylated form (IIo ).

Eighty-three syn mutants were obtained: 43 belong to nine complementation groups, 30 were left unassigned. Seventeen mutations were found in gene KIN28. Such a large number was expected from the genetic test itself since any mutation annihilating the function of Kin28p prevents the loss of the ADE3 plasmid carrying the KIN28 wild-type allele. Gene CCL1, which encodes a C-like cyclin, was affected 14 times, confirming that Ccl1p really interacts with Kin28p and strengthening the assertion of Kin28p being a CDK. CCL1 being an essential gene, it is somewhat surprising that CCL1 is almost as mutated as KIN28. The finding that syn mutations affect two genes whose products are involved in transcription, either as regulators of mRNA synthesis of particular sets of genes (SIN4) or as components of the basal transcription machinery associated with RNA polymerase II (RAD3), led us to search whether mutation kin28-ts3 could interfere with the activity of RNA polymerase II. The transcription of eight genes was studied (five of them previously chosen by Guzder et al. (1994) to identify the function of Rad3p). The steady-state level of their mRNA decreased drastically in a kin28-ts3 strain at restrictive temperature. Since these genes were chosen at random, Kin28p is most likely part of the basal transcription machinery. A kinase activity specific for the C-terminal repeat domain (CTD) of the Rpb1p subunit of RNA polymerase II has been shown to be associated with yeast general transcription factor H (TFIIH: previously termed as transcription factor b: Feaver et al., 1991). Since we have obtained evidence that KIN28 interacts genetically with RAD3, itself a component of this transcription complex, we can postulate that Kin28p, associated with Ccl1p, is the kinase

JMB—MS 566 Cyclin-dependent Kinase Kin28p and Transcription

associated with this factor. Thus, Kin28p should be a CTD kinase. Phosphorylation of the CTD of the Rpb1p subunit is thought to be an essential step of the transcription process: the unphosphorylated form enters the preinitiation complex and functions in promoter-proximal transcription, while the highly phosphorylated form sustains elongation (Drapkin & Reinberg, 1994; Usheva et al., 1992). Several kinases have been shown to phosphorylate the CTD in vitro (Dahmus, 1994), they include Ctk1p (Lee & Greenleaf, 1991) in yeast, Cdc2p (Cisek & Corden, 1989) in mouse cells, and MAP kinases (Dubois et al., 1994). We have shown that, in vivo, at restrictive temperature, the phosphorylation of the CTD is clearly reduced in mutant kin28-ts3. Intermediate states of phosphorylation which are still visible in Figure 3, could be explained in two ways. (1) The activity of the kin28-ts3 encoded protein is not completely blocked at 39°C (2) Other kinases phosphorylate the CTD. In support to the latter hypothesis, MAP kinases also appear involved in the in vivo CTD phosphorylation (Dubois et al., 1994). These results indicate that Kin28p is an essential component of the machinery carrying out the phosphorylation of the CTD. It may act either directly as an essential CTD kinase or indirectly as an activator of CTD kinases. Recently, Feaver et al. (1994) identified Kin28p as a subunit of transcription factor TFIIH, by biochemical and immunological approaches. Kin28p, together with two other proteins (p47 and p45; one of these proteins being certainly Ccl1p), constitute the TFIIK factor which associated with TFIIH and SSL2 = RAD25, form the holo-TFIIH factor (Svejstrup et al., 1994). In vitro, the holo-TFIIH factor has a CTD kinase activity (Feaver et al., 1994; Svejstrup et al., 1994). These data strongly reinforce the eventuality that Kin28p acts in vivo directly in the phosphorylation of the CTD. Comparisons with data bases show that Kin28p has its highest percentage of homology with the CDK-activating kinases of starfish, Xenopus and mammalian cells (Valay, 1994). Interestingly, Roy et al. (1994) have shown that the TFIIH-associated CTD-kinase of human cells is Mo15p. Micro-injection of antibodies which bind to Mo15p inhibited transcription. These data strongly suggest that Kin28p might be equivalent to Mo15p and be a CDK-activating kinase (CAK). However, no data are available, which back up this hypothesis (Solomon, 1994; Feaver et al., 1994; J.-G.V. et al., unpublished results). Factor TFIIH is required during nucleotide excision repair (NER) in yeast (Frieberg, 1994; Sweder, 1994; Drapkin et al., 1994). syn581 is a mutation in RAD3 which affects both reparation (UV sensitivity) and very likely transcription (synthetic lethality with kin28-ts3), at 26°C. We have asked whether, among the seven different kin28-ts mutants we have isolated, some of them could be UV-sensitive at 26°C (29°C being the restrictive temperature for five of the kin28-ts mutants (Valay, 1994). The kin28-ts mutants and the isonuclear wild-type strain were UV-irradiated. Their UV-sensitivities are similar

539 (data not shown), suggesting that either the mutations did not hit the domain(s), which would render Kin28p defective in nucleotide excision repair, or Kin28p is not involved in this process. Jiang & Stillman (1992) obtained results indicating that Sin4p acts as a negative regulator of transcription. Chen et al. (1993) have shown that Sin4p ( = Tsf3p) is a negative regulator of GAL1 and GAL10 promoters. Conversely, Sin4p appears to be a positive regulator of CTS1 (chitinase) (Jiang & Stillman, 1992). Jiang & Stillman have suggested that Sin4p could be neither a direct activator nor a repressor, but that it could effect transcription regulation through changes in chromatin structure, the Dsin4 null mutation leading to a decrease in the superhelical density of several plasmids. The behaviour of Sin4p can be compared with that of a negative regulator for the expression of gene HO, Sin1p = Stp2p, which is homologous to the chromatin-associated protein Hmg1p (Peterson et al., 1991). Furthermore, a SIN1 deletion relieves the INO1 transcription defect and cold-sensitive growth phenotype of strains whose CTD contains only ten heptamer repeats. It is recognized that chromatin structure plays a role in transcription initiation and elongation and that activation of transcription involves changes in its structure (Winston & Carlson, 1992; Kornbery & Lorch, 1992; Svaren & Ho¨rz, 1993). Our results, indicating that mutations in gene SIN4 are synthetic-lethal with mutation kin28-ts3, suggest that a functional relationship exists between the activity of Kin28p and chromatin states. Mutations in gene STI1 were found syntheticlethal with mutation kin28-ts3. When a wild-type copy of STI1 is borne on a multicopy vector, the in vivo transcription of the SSA4 heat shock mRNA is stimulated sevenfold (Nicolet & Craig, 1989). Moreover, Sti1p contains internal tetratricopeptide repeats (Boguski et al., 1990), in an arrangement resembling that of Ski3p, which is involved in RNA regulation (Sikorski et al., 1990). However, these data are not conclusive enough to decide whether Sti1p is a transcription factor. In chicken, a protein homologous to Sti1p, p60, is associated with Hsp90p and Hsp70p to form a complex which interacts with progesterone receptor (Smith et al., 1993). Judging from the abundance and widespread occurrence of this multiprotein complex, Hsp90p, Hsp70p and p60 probably function interactively in other systems as well (Smith et al., 1993). Hsp90p has been shown to interact with several protein kinases (Smith et al., 1993, and references therein). Recently, Aligue et al. (1994), have shown that the formation of active Wee1p protein kinase of Schizosaccharomyces pombe requires a physical interaction with Swo1p, an Hsp90p homolog. From these data we speculate that in S. cerevisiae Sti1p, in association with Hsp90p, establishes or stabilizes a particular conformation of Kin28p or of a component with which Kin28p interacts (e.g. Ccl1p, Rpb1p, subunits of TFIIH, . . .) or is required for the assembly of Kin28p in the transcription complex (Rutherford & Zuker, 1994; Jakob & Buchner, 1994).

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Figure 4. Genetic interactions between KIN28 and the syn genes RAD3, MCA28, SIN4, STI1, CDC37 and CCL1 are indicated by continuous lines. Genetic interactions which are putative or identified by other authors are represented by broken lines.

The exact function of Cdc37p at ‘‘Start’’ is unknown. Interestingly, cells carrying cdc28 and cdc37 temperature-sensitive mutations are barely viable at permissive temperature, indicating a functional interaction between these two gene products (Reed et al., 1985). On the other hand, several works show that the function of Cdc37p could be related to that of Hsp90. The expression of v-src in S. cerevisiae, which has no endogenous src gene, is lethal. However, this lethality is suppressed by a mutation in CDC37 or by reducing the level of Hsp82p, the yeast Hsp90 (Xu & Lindquist, 1993; Boschelli, 1993). Recently, it has been shown that two dominant mutations (named enhancers of sevenless) that reduce the frequency of the R7 photoreceptor neurons in Drosophila, in the presence of a barely functional sevenless protein (sevenless is a receptor protein tyrosine kinase), affect two genes whose products are homologous to yeast Cdc37p and to Hsp90p, respectively (Cutforth & Rubin, 1994). So, it is interesting to note the conjugate influences of Cdc37p and Hsp90p on the activity of these protein kinases. In conclusion, the interaction between Cdc37p and Kin28p suggests a possible connection between the activity of Kin28p and the cell cycle control.

The genetic interactions between KIN28 and the syn genes we have studied, as well as some of the putative interactions we have discussed of, are schematized in Figure 4. In summary, our in vivo results argue in favour of Kin28p being a cyclin-dependent kinase and show that it is necessary for the transcription of messenger RNAs. The facts that KIN28 interacts genetically with RAD3 and that the phosphorylation of the C-terminal repeat domain of the Rpb1p subunit of RNA polymerase II is affected in mutant kin28-ts3, strongly suggest that Kin28p is the kinase associated with factor TFIIH. Kin28p appears to be either an essential CTD kinase or an activator of CTD kinases. Our results are consistent with those obtained in vitro by Feaver et al. (1994). The genetic interactions between KIN28 and SIN4 or CDC37 suggest that the chromatin structure influences, directly or indirectly, the function of Kin28p and that a relationship does exist between the activity of Kin28p and the cell cycle control. Thus, Kin28p would be the second CDK after Pho85p (Espinoza et al., 1994; Measday et al., 1994) which might have a dual function.

Materials and Methods Strains and media Strains used for this study are described in Table 2. Complete medium: (YPD) 1% (w/v) yeast extract, 1% (w/v) Bactopeptone, 2% (w/v) glucose. G8 is complete medium with 8% (w/v) glucose. Synthetic complete medium (SC) is as described by Sherman (1991). SC (-LEU), SC (-URA) and SC (-LEU, -URA) are synthetic complete media lacking leucine, uracil or both, respectively. Plasmids pJG37 and pJG38 are vectors YEplac181 and YCplac33, respectively (Gietz & Sugino, 1988), bearing the 2.46 kb SpeI-PstI KIN28-containing fragment cloned at the XbaI and PstI sites of the polylinker. pJG72 is plasmid pJG37 bearing the 3.7 kb NheI-BamHI ADE3-containing fragment (Staben & Rabinowitz, 1986) ligated to SacI/BamHIdigested pJG37 (NheI and SacI restriction sites were blunt-ended using the Escherichia coli DNA polymerase I klenow fragment). YEp24-CDC28 was kindly provided by C. Mann. p 522 is YCplac33 bearing the 5.4 kb SalI-SacI

Table 2. Yeast strain genotypes Name GF260-2 GF262-2 GF309-72 GF310-131 FF18-358 JGV4 JGV105 GF314-17B GF314-26C SYN581

Genotype

Source

MATa leu2 trp1 ura3 lys2 MATa leu2 trp1 ura3 his3 MATa leu2 trp1 ura3 lys2 ade2 MATa leu2 trp1 ura3 lys2 ade2 ade3 MATa rad3-e5 his7-1 lys2-1 ade2 MATa leu2 trp1 ura3 his3 kin28-ts3 MATa leu2 trp1 ura3 his3 kin28-ts3 MATa leu2 trp1 ura3 ade2 ade3 lys2 kin28-ts3 MATa leu2 trp1 ura3 ade2 ade3 lys2 kin28-ts3 MATa leu2 trp1 ura3 ade2 ade3 lys2 kin28-ts3 syn581 (pJG72)

Simon et al. (1993) Simon et al. (1993) This work This work F. Fabre Valay et al. (1993) Valay et al. (1993) JGV4 × GF310-131 JGV4 × GF310-131 This work

All the strains used, strain FF18-358 excepted, are isonuclear.

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RAD3-containing fragment (Naumoski & Friedberg, 1992) cloned at the SacI and SalI sites of the polylinker. Screening strains used for the search of kin28-ts3 colethal mutations The ADE2 gene, borne by plasmid pASZ10 (Stotz & Linder, 1990), was deleted of its EcoRV-NdeI DNA fragment, yielding p361. Strain GF260-2 was then co-transformed (Rothstein, 1991) with BglII-cut p361 and with 0.5 mg of vector YEplac181 (Gietz & Sugino, 1988). One of the two red ade2 colonies that appeared among 7500 Leu+ transformants (named GF309-72) was sub-cloned on complete medium to lose vector YEplac181. The BamHI-NheI ADE3-containing fragment (Staben & Rabinowitz, 1986) was cloned into vector pTZ19R. A large part of the ADE3 coding sequence was deleted by removing the XhoI-HpaI DNA fragment, yielding p368. Strain GF309-72 was then co-transformed with BamHI-cut p368 and with 0.5 mg of vector YEplac181. Ten white ade2 ade3 colonies grew out of 10,000 Leu+ transformants; YEplac181 was lost from one of them, GF310-131, as described above. Let us recall that an ade3 haploid strain has a His− phenotype. Strains GF310-131 and kin28-ts3 were crossed. Diploids were selected and forced to sporulate. Two segregants, GF314-17B and GF314-26C, were transformed with plasmid pJG72. The resulting transformants were called GF314-17B (pJG72) and GF314-26C (pJG72). Colony-sectoring assay We have followed the method developed by Bender & Pringle (1991). Strains GF314-17B (pJG72) and GF314-26C (pJG72) were subcloned on SC (-LEU). One colony was dispersed in water. 2.5 × 105 cells in 10 ml of water were UV irradiated in a glass Petri dish to approximately 10% survival. One-hundred G8 plates were each plated with 0.1 ml of the irradiated suspension and then incubated for five to six days at 26°C. Uniformly red colonies were streaked on G8; after three to four days at 26°C, individual colonies were restreaked on G8. Only clones still giving red colonies were studied further. To test whether red colonies harbour synthetic lethal mutations (syn) and do not result from chromosomal integration of plasmid pJG72 or conversion of the deleted ade3 gene, mutants were transformed with either plasmid pJG38 (KIN28) or plasmid YEplac33 (used as a control). Four colonies from each transformation were streaked on G8 plates. Mutants giving sectored colonies with pJG38 and only red ones with Yeplac33 were studied further. For complementation studies, GF314-17B (pJG72) and GF314-26C (pJG72)-derived mutants were mated on YPD; after four days at 26°C, two samples of diploids were streaked on G8 plates. If both mutations are allelic every colony must be red, whereas the presence of sectored colonies indicates that both mutations belong to different complementation groups. Screening of thermosensitive synthetic lethal mutations syn mutants were grown to stationary phase in YPD medium at 24°C. Five microliters of these cultures were dropped onto YPD plates, in duplicate. Plates were left three days at 24°C for one set, at 37°C for the other. Several syn mutants appeared to be thermosensitive. To test whether the thermosensitivity is due to their synthetic

lethal mutations, Mat a and Mat a syn-ts mutants were mated with GF314-26C and GF314-17B, respectively, on YPD. Diploids were selected on SC plates lacking lysine, histidine and leucine. After sporulation, the thermosensitivity and synthetic lethality of segregants were analysed. Isolation of SYN genes Thermosensitive synthetic lethal mutants were transformed with a wild-type genomic library built with the centromeric vector YCp50, harbouring the URA3 selective marker (Rose et al., 1987). Vector YEplac33 was used as a control. Transformants were selected on SC (-LEU, -URA) plates at 24°C, then replica-plated onto YPD plates which were left three days at 37°C. Master-plates were made by transplanting thermoresistant transformants onto SC (-LEU, -URA) and YPD plates, at 24°C. To differentiate thermoresistant transformants from thermoresistant revertants, we performed the following test. Samples from the YPD master-plates were sub-cloned on YPD plates which were left four days at 24°C, to allow the loss of plasmids, then were replica-plated on SC (-LEU) and SC (-URA) plates which were incubated at 24°C and on YPD plates which were incubated at 37°C. Thermoresistant transformants, which turned thermosensitive in losing either the pJG72 plasmid or the YCp50-derived plasmid, and remained thermoresistant in keeping both plasmids, were studied further. The YCp50-derived plasmids carried by the thermoresistant transformants were transferred in E. coli strain MC1066 by electroporation (Dower et al., 1988). They were amplified and used to transform the corresponding yeast thermosensitive lethal mutants. Plasmids conferring thermoresistance and giving sectored colonies were analysed by a one-step disruption method using a Mini-Mu derivative to localise the SYN genes. The E. coli strains used were MC4100::(Mucts)::(MudIIPR13) and M8820 (Richaud et al., 1987; Diagnan-Fornier & BolotinFukuhara, 1988). The Mini-Mu junctions were sequenced on both sides by the dideoxy chain-termination method with the synthetic oligonucleotides as described by Dang et al. (1994). Screening of UV-sensitive synthetic lethal mutants syn mutants were grown to stationary phase in YPD medium at 24°C and 2000 cells were dropped onto YPD plates. They were UV-irradiated with the following doses: 0, 25, 50, 100 or 150 J/m2, and left three days at 27°C (five syn mutants, particularly syn581, appeared clearly UV-sensitive). Isolation and analysis of RNA Strains kin28-ts3 and GF262-2 were grown at 24°C in 250 ml of YPD medium to an A600 of 1.0 to 1.2. Seven samples (25 ml each) were removed from each culture, transferred into 100 ml Erlenmeyers and shaken immediately in a 38°C water bath during 0, 15, 30, 45, 60, 120 and 180 minutes (Qiu et al., 1993). After incubation, the flasks were left two to three minutes on ice. Cells were centrifuged, washed in cold sterile water and centrifuged again. Cell pellets were frozen at −80°C. Total cellular RNA was prepared as follows: cell pellets were resuspended in 400 ml of RNA extraction buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 5% (w/v) SDS) then transferred to 2.0 ml microfuge tubes containing 300 ml of glass beads and 500 ml of PCI (phenol/ chloroform/isoamylic alcohol (50:50:1, by vol.). The tubes

JMB—MS 566 542 were vortexed for two minutes at room temperature. The phases were separated by centrifugation and the upper phases were transferred to fresh microfuge tubes containing 700 ml of PCI. The phenol phases were re-extracted with 400 ml of RNA extraction buffer. After centrifugation the upper phases were pooled with the former ones and extracted four more times with PCI. RNA was precipitated with ethanol, treated with RNase-free DNAase I (Worthington) and quantified by spectro-photometry. An equal amount of RNA (9 to 10 mg) from each sample was denatured, submitted to electrophoresis and transferred to nitrocellulose paper as described by Thomas (1983). The DNA fragments used as hydridization probes, borne in vector pTZ19 or synthesized by PCR (RAD23, TRP3, ACT1, CDC9, MET19), were 32P-labelled by ‘‘oligo-labelling’’ (Pharmacia). Western blot analysis The protocol to examine the phosphorylation state of Rpb1p was adapted from previous studies (Dubois et al., 1994). In brief, yeast cultures were grown in YPD medium at 24°C to an A600 of 1.0 to 1.2. They were transferred at 39°C, then 5 ml samples were removed at 0, 10, 20, and 30 minutes, poured into 50 ml of ice-cold water, centrifuged, resuspended in 200 ml of ethanol and placed on solid CO2 . Cells were disrupted as described by Lee & Greenleaf (1991) by vortexing with 100 ml of glass beads (three times for one minute with one minute intervals on solid CO2 ). The precipitate was dried, resuspended in 500 ml of Laemmli buffer (60 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% (v/v) glycerol, 1% (v/v) 2-mercaptoethanol and 0.002% (w/v) bromophenol blue) and heated for five minutes at 90°C. Samples were electrophoresed in 5% (w/v) polyacrylamide/SDS gels. Proteins in the gel were electrotransferred onto nitrocellulose membranes (0.45 mm) (Schleicher and Schuell). The membranes were blocked in Tris-buffered saline (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.2% (v/v) Tween 20) containing 5% (w/v) non-fat dry milk and incubated for one hour in blocking solution with a 1/5000 dilution of the monoclonal antibody 8WG16 (which is directed against the CTD and was kindly provided by Dr N. E. Thompson (Thompson et al., 1990)). After washing in Tris-buffered saline, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1/7500) in blocking solution for one hour. The blots were visualized using chemiluminescence (Renaissance, DuPont NEN).

Acknowledgements We are grateful to Carl Mann, Francis Fabre, Roland Chanet, Philippe Reisdorf, Bertrand Diagnan-Fornier, Bertrand Se´raphin, Franc¸ois Lacroute for strains, plasmids and useful suggestions, and Agnes Leray for excellent secretarial assistance. This work was supported by Association pour la Recherche sur le Cancer (contract number, 6087) and by La Ligue Nationale contre le Cancer.

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Edited by M. Yaniv (Received 13 February 1995; accepted 24 March 1995)