Regulation of RNA processing and transport by a nuclear guanine ...

The EMBO Joumal vol.12 no.7 pp.2929-2937, 1993

Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily Tatsuhiko Kadowaki, David Goldfarb1, Lynn M.Spitz1, Alan M.Tartakoff2 and Mutsuhito Ohno3 Institute of Pathology, Case Westem Reserve University, 2085 Adelbert Road, Cleveland, OH 44106, 'Department of Biology, University of Rochester, Rochester, NY 14627, USA and 3Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan 2Corresponding author Communicated by I.Mattaj

The RCCI gene of mammals encodes a guanine nucleotide release protein (GNRP). RCC1 and a homolog in Saccharomyces cerevisiae MTR1/PRP20/SRM1) have previously been implicated in control of mRNA metabolism and export from the nucleus. We here demonstrate that a temperature-sensitive fission yeast mutant which has a mutation in a homologous gene, and two of three additional (mtrl/prp201srml) mutants accumulate nuclear poly(A)+ RNA at 37°C. In S.cerevisiae, maturation of rRNA and tRNA is also inhibited at 37°C. Nevertheless, studies with the corresponding BHK-21 cell mutant indicate that protein import into the nucleus continues. MTRI homologs regulate RNA processing at a point which is distinct from their regulation of chromosome condensation since: (i) poly(A)+ RNA accumulation in the fission yeast mutant precedes chromosome condensation, and (ii) unlike chromosome condensation, accumulation of nuclear poly(A) + RNA does not require p34cdc28 kinase activation or protein synthesis. Moreover, experiments involving inhibition of DNA synthesis indicate that the S.cerevisiae homolog does not govern cell cycle checkpoint control. Since RCClp acts as GNRP for Ran, a small nuclear GTPase of the ras superfamily, we have identified two homologs of Ran in S.cerevisiae (CNR1 and CNR2). Only CNRI is essential, but both code for proteins extremely similar to Ran and can suppress mtrl mutations in allele-specific fashion. Thus, MTR1 and its homologs appear to act as GNRPs for a family of conserved GTPases in controlling RNA metabolism and transport. Their role in governing checkpoint control appears to be restricted to higher eukaryotes. Key words: cell cycle/GTPase/piml/RCCl/RNA processing/tsBN2

Introduction We have initiated a genetic approach to the study of mRNA export by identifying conditional temperature-sensitive (ts) mutants of Saccharomyces cerevisiae which accumulate poly(A)+ RNA in the nucleus at the restrictive temperature (Kadowaki et al., 1992). For the first of these mutants, mtrl-1, the poly(A)+ RNA which accumulates at 37°C is distributed through much of the nucleoplasm, as judged by light microscopic in situ hybridization. This intranuclear pool Oxford University Press

of poly(A)+ RNA is remarkably stable and much of it can be exported when the temperature is reduced to 23°C. We now report the cloning of the MTRM gene and identification of point mutations at the MTRJ locus. [Although this gene is identical to PRP20 and SRMI (see below) we refer to it simply as MMRJ, both for simplicity and because its role in mRNA transport is more broadly established than its other roles.] We have also characterized RNA processing and cell cycle progression in mtrl mutants, investigated the consequences of ts mutations of MTRM homologs and identified GTPases of the Ras family which appear to interact with Mtrlp. Previous studies have shown that Mtrlp and/or its homologs in other species (RCC1 in hamsters, pim 1 in Schizosaccharomyces pombe) are nuclear proteins (Fleischmann et al., 1991; Seino et al., 1992; Amberg et al., 1993), associate with chromatin (Ohtsubo et al., 1989; Bischoff and Ponstingl, 1991a; Seino et al., 1992) have guanine nucleotide release protein (GNRP) activity toward a small Ras-like GTPase, Ran (in mammals), (Bischoff and Ponstingl, 1991a,b; Ren et al., 1993), affect several aspects of mRNA metabolism (Aebi et al., 1990; Forrester et al., 1992; Amberg et al., 1993), and control DNA synthesis (Nishimoto et al., 1978; Dasso et al., 1992) and chromosome condensation and/or cell cycle checkpoint control (Nishimoto et al., 1978; Matsumoto and Beach, 1991; Nishitani et al., 1991; Seino et al., 1992). In S.pombe, a ts mutation in the RCCJ homolog, piml +, can be suppressed by the Ran homolog, spil +. Thus, pimI appears to interact with spil, much as the RCC 1 product interacts with Ran.

Results Identification of the MTR1 gene and point mutations We have isolated three different recessive mutant alleles of the MTRI gene (mtrl-l, 1-2 and 1-3) (Kadowaki et al., 1992). To clone the MTRI gene, YTK102(mtrl-1) was transformed with a YCp5O yeast genomic DNA library. The complementing DNA fragment was identical to the previously identified unique SRMIIPRP20 gene, which is essential for vegetative growth (Clark and Sprague, 1989; Aebi et al., 1990). The S. cerevisiae MTR1 protein shows significant similarity to mammalian, Drosophila and Spombe homologs and includes seven glycine-rich approximate repeats (Ohtsubo et al., 1987; Frasch, 1991; Matsumoto and Beach, 1991). To understand the structure -function relationships of this protein, the sites of mutation in mtrl-1, 1-2, 1-3 and srml-l have been identified. In mtrl-l glycine 338 is changed to aspartic acid, in mtrl-2 glycine 109 is changed to serine, in mtrl -3, as in prp2O-4 (Amberg et al., 1993), glycine 464 is changed to aspartic acid, and in srml-l glycine 117 is changed to aspartate. In prp2O-1 glycine 457 is changed to glutamic acid (Aebi et al., 1990; Fleischmann et al., 1991). The serine to phenylalanine substitution in the homolog in the BHK-21 cell mutant, tsBN2, is at a position

2929

T.Kadowaki et al.

A

B

Cmqo

Fig. 1. Distribution of poly(A)+ RNA in S.cerevisiae. (A) The indicated ts strains were incubated at 37°C for 3 h and then fixed and processed for in situ hybridization using oligo(dT)-biotin and FITC-avidin. As illustrated, in all alleles except snnl-l, a conspicuous signal (arrowheads) is seen. No nuclear intensification was seen when the strains were examined after growth at 23°C. For panels 1-1, 1-2, 1-3 and srm, the fluorescent signal coincides with DNA (DAPI) staining of nuclei. wt, wild-type cells; 1-1, mtrl-J; 1-2, mtrl-2; 1-3, mtrl-3; srm, srml-1. (B) When strain YTK11O, in which MTRJ expression is under galactose control, is shifted from 2% raffinose-0.0075% galactose to 2% glucose medium, growth stops. As shown (arrowheads), a clear nuclear accumulation of poly(A)+ RNA is seen after 24 h. (C) Poly(A)+ RNA (P) and DNA (D) distributions in single mutants and in mtrl-l prtl-1, mtrl-l cdc28-1 and mtrl-l cdc28-JN double mutants. Each cell type was incubated 3 h at 37-38°C and then processed. 1, mtrl-l; 2, prtl-l; 3, mtrl-l prtl-1; 4, cdc28-1; 5, mtrl-l cdc28-1; 6, cdc28-JN; 7, mtrl-l cdc28-JN. Clearly neither CDC28 activation nor PRT1 function is required to give a strong nuclear in situ hybridization signal (arrowheads). Cells maintained at 23°C showed uniform cytoplasmic and nuclear fluorescence. In the in situ hybridization images, relatively dark areas (narrow arrowheads) correspond to vacuoles.

corresponding to residue 290 in the yeast sequence (see Ohtsubo et al., 1991). Distribution of poly(A)J RNA in S.cerevisiae The mtrl-l and several prp2O mutants, which have distinct point mutations, accumulate poly(A) + in the nucleus at

2930

37°C (Forrester et al., 1992; Kadowaki et al., 1992; Amberg et al., 1993). As shown in Figure IA, mtrl-2 and mtrl-3 also exhibit nuclear poly(A)+ RNA accumulation, when analyzed by in situ hybridization. In contrast, the cytoplasmic fluorescent signal of srml-l remains uniform and strong at 37°C and nuclear staining is not increased.

Yeast RNA transport and GTPases

A

mtr 0

5 10

B

Vt 0

5)

10

9 64

w

25C

m

r

w

32

mr

35s

-32 -27 -25

5.8s

S p

w

t

Fig. 2. rRNA and tRNA processing. (A) rRNA. Wild-type (YIK100) and mtrl-l (YTK102) strains were pre-incubated at 37°C for 30 min and then labeled for 3 min with [3H]methylmethionine followed by a 0, 5 or 10 min chase. The labeled RNA was electrophoresed on a 1% formaldehydeagarose gel. The first three and second three lanes show RNA isolated from mtrl-l and wt, respectively. The first and fourth lanes are samples withdrawn at the end of the pulse. The second and fifth lanes: 5 min chase. The third and sixth lanes: 10 min chase. In the mutant, by comparison with wild-type, all processing steps appear to be slowed. (B) tRNA. Wild type (YTK100) (w), rntrl-l (YTK102) (m) and rnal-I (EElb rnal-1) (r) strains were preincubated at 37°C for 30 min and then labeled for 10 min with [3H]uridine. The labeled RNA was electrophoresed on a 10% polyacrylamide-8 M urea gel which was fluorographed. The two mutants show similar accumulation of tRNA precursors (p) and a weakening of label of 5.8S RNA. The wt and mtrl-l cells were transformed with pRS316 (Sikorski and Hieter, 1989) for this experiment to make them URA3+. t, mature tRNA.

To define the consequences of total loss of function of this gene, we have created a yeast strain in which the MTRJ gene

is under galactose control. During glucose incubation cell growth ceases at 12 h, cells enlarge and aggregate and both large budded and unbudded cells come to dominate the population. As shown in Figure lB an obvious accumulation of poly(A)+ RNA in the nucleus is observed in 40-50% of the cells. Thus, depletion of Mtrlp appears to block mRNA export. Overexpression of Mtrlp by transfer to 2% galactose is toxic and does not arrest cells at a single point in the cell cycle. It does reduce the titer of poly(A)+ RNA throughout the cell (not shown). Protein synthesis and p34cdc28 kinase activation are not required for accumulation of nuclear poly(A)+ RNA in mtrl-l To check the possible involvement of p34cdc28 kinase activation and protein synthesis in the mRNA export block

in mtrl-1, we constructed mtrl-l cdc28-1, mtrl-l cdc28-lN and mtrl-l prtl-l double mutants. cdc28-1 arrests at GI at 37°C while cdc28-1N arrests in G2 at 37°C (Surana et al., 1991). prtl-l is ts for initiation of translation and exhibits complete breakdown of polysomes within 2 min at 37°C (Hartwell and McLaughlin, 1969). As shown in Figure IC, the double mutants exhibit nuclear poly(A) + RNA accumulation at 37°C. Thus, the mRNA export block does not require p34cdc28 kinase activation or protein synthesis. In this sense, the effects of mutation of the MTRJ gene on RNA processing appear to be more direct than the effects of mutation of RCCJ or piml + on chromosome condensa-

tion (Nishimoto et al., 1981; Matsumoto and Beach 1991; Nishitani et al., 1991). Cell cycle arrest with hydroxyurea, factor or nocodazole does not lead to nuclear accumulation a

of poly(A)+ RNA (not shown). RNA processing in mtrl- 1 Previous studies have shown that m7G cap methylation of poly(A)+ RNA occurs in mtrl-l at 370C and that the poly(A) tail of poly(A)+ RNA in mtrl-l and prp2O-1 at 37°C is longer than in wild-type cells (Forrester et al., 1992; Kadowaki et al., 1992). This latter result is reminiscent of

rnal-J (Piper and Aamand, 1989) and is consistent with the fact that progressive shortening of the poly(A) tail of mRNA is thought to occur in the cytoplasm (Sachs, 1990). The synthesis and processing of rRNA in mtrl-l was analyzed by labeling with [3H]methylmethionine. As shown in Figure 2A, pulse-labeling of rRNA in mtrl-l is severely reduced after 30 min at 37°C and processing of 35S preRNA to 27S and 20S rRNA is very slow, judging from the persistence of 35S pre-rRNA throughout the chase period. Furthermore, mature 25S and 18S rRNA do not accumulate during the chase period, indicating a slowing of processing from 27S to 25S and 20S to 18S rRNA. Comparable defects were observed in [3H]uridine labeling experiments (not shown) and have previously been observed in the rnal mutant (Traglia et al., 1989). [3H]uridine labeled RNA in mtrl-l was also analyzed to monitor the synthesis and processing of small RNAs. Labeling of tRNA continues, but synthesis of 5.8S rRNA and 5S rRNA is inhibited at 37°C. The processing of pre-tRNA in mtrl-l is also inhibited, as in rnal-1 at 37°C (Figure 2B). 2931

T.Kadowaki et al.

Cell cycle progression and checkpoint control in mtrl mutants The srml-l mutant was originally characterized as transcribing mating factor-induced genes at the restrictive temperature in the absence of mating factor and accumulating in G1 (when in a AL4Tc, ste3- background) (Clark and Sprague, 1989). We examined whether other mtrl alleles also show cell cycle arrest at 33 or 37°C. No specific cell cycle arrest is seen at 37°C, but the fraction of unbudded cells increases at 33°C (to 74-87%) in mtrl-1, 1-2, 1-3 and prp20-1 mutants. Considering previous observations on the hamster BHK-21 and S.pombe homologs, a key question is whether incubation of mtrl mutants at 37°C may lead to loss of checkpoint control. We have evaluated this possibility by incubating each mutant with or without hydroxyurea for 6 h at 37°C and then estimating survival upon plating at 25°C. Unlike strains that are hypersensitive to hydroxyurea and/or radiation

damage (Murray, 1992), the survival of each of the five mtrl strains is reduced only -2-fold due to the presence of hydroxyurea. Similar results were obtained with a secl mutant incubated with or without hydroxyurea (not shown). Furthermore, when mtrl strains are incubated with hydroxyurea for as long as 6 h at 37°C, they simply accumulate with large buds. There is no morphological indication that they continue through the cell cycle. Thus, there is no evidence that the S. cerevisiae MTRI gene governs checkpoint control. Since mitotic chromosome condensation is not visible in S. cerevisiae, chromosome condensation itself cannot be evaluated. Analysis of mammalian and S.pombe mutants The BHK-21 cell tsBN2 mutant has a point mutation in RCCI (Uchida et al., 1990). It was originally identified as a mutant which arrested in GI and ceased DNA synthesis at 39.50C (Nishimoto et al., 1978). Additionally, when synchronized at G1/S at 33°C, many cells undergo rapid chromosome condensation at 39.5°C. The S.pombe piml-46 mutant also exhibits chromosome condensation at 37°C, even in the presence of hydroxyurea (Matsumoto and Beach, 1991). Since ts mutations in the S. cerevisiae homolog of RCCI and piml + cause nuclear accumulation of poly(A) + RNA, we asked whether the RCCI mutant, tsBN2, and piml-46 also accumulate nuclear poly(A)+ RNA at the restrictive temperature. Consistent with a recent report (Amberg et al., 1993), tsBN2 cells do show a striking nuclear accumulation of poly(A)+ RNA when incubated at 39.5°C (Figure 3A). The defect does not block protein import, judging from experiments in which cells preincubated at 39.5°C for 6 h were micro-injected (cytoplasmically) with fluorescent BSA derivatized with the Ela nuclear localization signal: the cytoplasmic tracer moves to the nucleus with kinetics similar to 33°C controls and BHK-21 cells at 33 or 39.5°C (Figure 3B). In wild-type S.pombe at 25 or 37°C and in piml-46 at 25 0C, the in situ hybridization signal is somewhat punctate and especially bright in the cytoplasm (Figure 4). In most piml-46 cells after 2 h at 370C one or two bright regions are visible in the nucleus. The poly(A)+ RNA signal and DAPI signal do not always overlap, suggesting that poly(A) + RNA may preferentially accumulate in a chromatin-poor region of nucleus. A comparable nuclear signal is seen when the 37°C incubation includes cycloheximide (not shown). When piml -46 is incubated for

2932

I-i}

Ct

o,

_

-LI

0_.

OLa

Fig. 3. Export and import across the nuclear envelope in the BHK-21 mutant, tsBN2. (A) Poly(A)+ RNA distribution mn BHK-21 and tsBN2 cells. Both BHK-21 (WT) and tsBN2 (TS) were incubated for 12 h at 33.5 or 39.50C and then fixed and processed as in Figure 1. In tsBN2 the accumulation of poly(A) + RNA in the nucleus is striking at 39.50C. Similar results were obtained with tsBN2 presynchronized at GI/S (not shown). (B) Protein import into the nucleus. Both BHK-21 (WT) and tsBN2 (TS) were incubated for 6 h at 33.5 or 39.50C before injection of fluorescent p(Ela)BSA. They were photographed after 30 min at the indicated temperatures. In all cases, the fluorescent were obtained after 5

protein has moved to the nucleus. Similar results min post-injection (not shown).

longer at 370C, chromosome condensation is seen [.15 % at 3 h, 50% at 3.5 h (Matsumoto and Beach, 1991)]. For example, after 4 h at 370C, most cells have condensed chromosomes and a septum. These cells have lost much of their nuclear poly(A)+ RNA signal. One of the rare cells in Figure 4 (arrow) that still ethibits a nuclear signal has non-condensed chromatin. Identificatio of S.c To detect a Ran/spil + gene homolog, we used degenerate primers for PCR amplification of S. cerevisiae genomic DNA

Yeast RNA transport and GTPases 8 V. .L. L .47 CNR1 MSAPAAN-GEVPTFKLVLVG0GKIrFVKRHLTTGEFEKY.IATIGVEVH 50

MAA4GEPQVQ .................

Ran

Spil+

COR2 wtl

Ran

Spil+

MAQPQN.

....

Q.NA

.

.51 G-2

G-1 ..V.H ..R.P.K.N . F .... L. ..H.H ... C.N ....

QA.A . ........ V

LG.G .........

.

98 97

aCRJ PLSFYTNFGEIKFDVWTAGQEKFGGLRDGYYINAQCAIIMFDVTSRITY 100 . . . 101 ... aR2*..... .

.

.

G-3

Ran

I.DS.

.... N.H .

S.V .148

A .147 Spil+ ... .H.W. CNR1 KVPNWHRDLVRVCENIPIVLCGLKMDVKERKVKAKTITFHRKKNLQYYD 150

QVR2

.. 151

G-4 Ran.

spil

G-

M .... VM. PA.AA. .E 198 I.D . V .N .Q. ..LA... 197

. ...

'2mi ISZKSNYNFEKPFLWLARKLAGNPQLEFVASPALAPPEVQVDEQLMQQYQ H... .

200 201

5

ts

Ran

HDLEV.QNPA .

Spil+

..

OMRI

CMR2

.NE.A.M

..

..... 216 216

QEMEQATALPLPDEDDADL 219 .. D... 220

Fig. 5. Amino acid sequences of CNR1, CNR2, spil and Ran products. The sequences are compared with Cnrlp. Dots indicate identity and dashes indicate gaps. The underlined segments (G-1 -G-5) are the domains which are thought to interact with guanine nucleotides (Boume et al., 1991). Fig. 4. Poly(A)+ RNA distribution and chromosome condensation in S.pombe wild-type and piml-46 mutant cells. Top: wild-type (wt) cells were fixed and processed after 0 or 2 h at 37°C. The cytoplasmic signal is often punctate. Nuclei are fainter than cytoplasm. Bottom: piml-46 (ts) cells were fixed and processed after 0, 2 or 4 h at 37°C. Nuclear poly(A)+ RNA is easily detected at 2 h, i.e. before chromosome condensation. At 4 h, when chromosome condensation is evident for many cells, the nuclear FITC signal persists only in those cells in which condensation has not occurred (arrow).

and obtained a single major 190 bp product. Upon subcloning, two sequences were found. Probing of a yeast genomic Southern blot with these sequences revealed that they correspond to two different genes, designated CNRJ and CNR2 (Conserved Nuclear Ras homologs 1 and 2). When a YCp5O yeast genomic library was screened with the two probes, full length CNRJ and CNR2 genes were detected (Figure 5). The two gene products clearly belong to the Ras superfamily, including the G-1 to G-5 motifs which bind GTP (Bourne et al., 1991). They resemble human Ran and S.pombe spil more closely than yeast or human Ras, Ypt or Rho GTPases. They have calculated molecular weights of 24 974 and 24 794 Daltons, are highly homologous in DNA sequence (83.3% homology) and nearly identical in protein sequence. Neither has an obvious nuclear localization signal (Dingwall and Laskey, 1991) or lipid addition signal (Magee and Newman, 1992). Probing of a chromosomal blot shows that CNRl and CNR2 are on distinct chromosomes (not shown). To test the importance of these genes for viability, we attempted disruption in wild-type cells. Haploid CNR2 disruptants grew well and pairs of CNR2 disruptants mate to produce viable diploids. Since in S*pombe, the copy number of spil + is critical for chromosome stability (Matsumoto and Beach, 1991), we have used a sectoring assay (Koshland et al., 1985) to evaluate stability of a centromeric plasmid in a haploid CNR2 disruptant. No increase in plasmid loss was seen (not shown).

Several experiments indicate that

CNRJ is essential. (i)

All attempts to disrupt CNRl in a haploid did not yield viable

disruptants. (ii) When one copy of CNRl was disrupted with a HIS3 marker in a diploid, the transformant (YMO106) grew, sporulated (poorly) and dissection of 14 asci having three or four spores yielded no his+ segregants. (iii) When CNRJ on a centromeric URA3+ plasmid was introduced into YM0106, the transformant (his+ ura+) sporulated well, yielded his+ ura+ spores and all attempts to obtain his+ ura- colonies with 5-FOA failed (Sikorski and Boeke, 1991). (iv) A haploid CNRl disruptant which carries CNRI under GAL] control on a plasmid grew in galactose medium, but not in glucose medium. In parallel experiments, CNR2 on either a centromeric plasmid or a 2 A plasmid rescues the growth defect of CNRI-disrupted haploid cells. To learn whether Cnrlp and/or Cnr2p may interact with Mtrlp, CNRI and CNR2 genes were cloned individually into either centromeric or 2 , plasmids and introduced into the five mtrl mutants. As illustrated in Figure 6 and sununarized in Table I, several mutations near the C-terminus of the protein [mtrl-l (residue 338), prp20-1 (residue 457) and, to a lesser extent, mtrl-3 (residue 464)], were suppressed by CNRl or CNR2 at 30/34°C, while mutations srml-l (residue 117) and mtrl-2 (residue 109) which are much closer to the N-terminus, were not suppressed.

Discussion Several observations indicate that Mtrlp is involved in mRNA export with some specificity: (i) poly(A)+ RNA accumulates in the nucleus ir1 mtrl-1, 1-2, 1-3 and prp20 mutants at 37°C, upon Mtrlp depletion and in related mutants in two other species, and (ii) poly(A)+ accumulation in mtrl-l does not require protein synthesis or p34cdc28 function. Nevertheless, the MTRI gene product clearly is important for multiple nucleoplasmic events.

2933

T.Kadowaki et al.

Since RCC1 associates with chromatin, and since elimination of RCC1 from Xenopus egg extracts blocks formation of DNA replication complexes, but not DNA elongation per se, RCC1 may be part of or regulate an abundant structural unit in the nucleoplasm. Such a structural unit may also interact with components of the transcriptional machinery, or equip polyadenylated RNA with factors which are essential for their transport. The importance of Mtrlp for rRNA and tRNA processing may reflect comparable dependence on this structural unit.

Alternatively, the mRNA export lesion might be primary and lead to progressive depletion of nuclear proteins which are required for multiple events. For example, it is conceivable that mere depletion of critical cytoplasmic RNAs at 37 -39.5°C in piml -46 and tsBN2 leads to activation of p34cdc2 kinase and chromosome condensation. Arguing against this possibility is the observation that G1/S synchronized BHK-21 cells treated at 39.5°C with actinomycin D or a-amanitin do not show chromosome condensation (unpublished observation). Moreover, the

Fig. 6. Complementation of growth of mtrl mutants by expression of CNRI or CNR2 at 30 and 34°C. Yeast strains. (A) mtrl-l, (B) mtrl-2, (C) mtrl-3, (D) prp2O-l, (E) snnl-l. Plasmids: a, pRS316; b, YEpCNR2; c, pRSCNR2; d, YEpCNRl; e, YEpCNR14.1; f, pRSCNRl. Plasmids YEpCNRI4.1 and YEpCNRI differ only in the CNRI 5' untranslated region. The pRS plasmids are centromeric and YEp plasmids are high copy. Specific suppression of mtrl-l, prp2O-l and mtrl-3 is seen at 30 and 34°C. No growth of the transformants is seen at 37°C. 34°C is restrictive for both growth and poly(A)+ RNA export for mtrl-1.

2934

Yeast RNA transport and GTPases

observation that premature chromosome condensation in tsBN2 does not absolutely require RNA synthesis (Nishimoto et al., 1981) argues against the possibility that nuclear accumulation of high levels of poly(A)+ RNA itself causes cell cycle progression. Therefore it is most likely that, in those organisms in which mutations of MTRJ homologs affect chromosome condensation, these effects are not mediated by gross alteration of RNA metabolism. Chromosome condensation in tsBN2 and piml-46 at 37-39.5°C requires protein synthesis and (in S.pombe at least) p34cdc2 kinase activation (Nishimoto et al., 1981; Matsumoto and Beach, 1991; Nishitani et al., 1991), unlike Table I. Suppression of mtrl mutants by CNR genes

300C

340C prp

srm

_

_

_

-

+-

++ + ++

+-

++

-

++

-

1-1

1-2 1-3

prp

srm

1-1

1-2 1-3

C cenA cenB 2yA

++ +++

-

+

_ -

-

+_-

-

-

-

++

+++-

++

-

++ +++

++

-

24A*

++ ++

-

++ ++

++ ++

+ +-

++ ++

-

+-

2AB

-

-

-, no suppression; + -, weak suppression; +, medium suppression;

+ +, strong suppression; 1-1, mtrl-l; 1-2, mtrl-2; 1-3, mtrl-3; prp, prp20-1; srm, srml-l; C, control transfornants (pRS316); cenA, CNRl gene on a centromeric plasmid pRS316; cenB, CNR2 gene on pRS316; 21tA, CNRI gene on a 2it plasmid YEp24; 2/AA*, the same as 2yA but contains extra 3' non-coding sequence of CNRJ; 2AB, CNR2 gene on YEp2.

the nuclear accumulation of poly(A)+ RNA. We therefore conclude that the consequences of loss of function of MTR homologs for RNA processing and transport are relatively direct. Also supporting these considerations is our observation that, in piml-46 at 37°C, accumulation of poly(A) + RNA in the nucleus definitely precedes chromosome condensation and p34cdc2 kinase activation (Matsumoto and Beach, 1991). Furthermore, since in S. cerevisiae (and apparently in S.pombe; S.Sazer, personal communication) loss of MTRI (or piml +) function does not cause loss of checkpoint control [or kinase activation (Clark et al., 1991)], the functions of this gene, although affecting several classes of RNA, appear more limited than for its homolog in animal cells. RCC1 promotes guanine nucleotide exchange by a small GTPase of the ras superfamily, Ran (Bischoff and Ponstingl, 1991a,b; Ren et al., 1993), which is highly homologous to spil, the extragenic suppressor ofpiml-46 (Matsumoto and Beach, 1991). The S. cerevisiae homologs that we have identified, CNRI and CNR2, encode products which are extremely similar to the products of spil/Ran genes and like spil and Ran, they appear to interact with the corresponding GNRP. Several considerations argue that CNRJ and CNR2 products have similar activities and that CNRl is simply more highly expressed than CNR2: (i) these two proteins are 97.7% identical in sequence; (ii) a CNRI disruptant can be rescued by increased copy number of CNR2; (iii) a centromeric CNRI plasmid suppresses mtrl mutants better than centromeric CNR2 (Figure 6); (iv) the codon bias index (Bennetzen and Hall, 1982), which correlates with protein

Table II. Yeast strains

S.cerevisiae

Strain

Source

Relevant genotype

SYI 115

EElb(rnal-l)

G.Sprague M.Clark Yeast Genetic Stock Center Yeast Genetic Stock Center A.Hopper

cdc28-1N

B.Futcher

YTK100

This This This This This This This

study study study study study study study

YMO1O1 YMO102 YMO103 YMO104 YMO106

This This This This This This This This

study study study study study study study study

MATa leu2-3, 112 ura3 trpl his4 srml-l MATa ura3-52 ade2-101 his3 A200 prp2O-1 MATa adel ade2 ural his7 lys2 tyri gall prtl-l MATa adel ade2 ural his7 lys2 tyrl gall leu2 cdc28-1 MA Ta rnhl:: URA3 ura3-52 tyrl his4 his7 adel rnal-l GalMATa ade2-1 trpl-I can1-100 leu2-3,112 his3-11,15 ura3 GAL [psi+] cdc28-JN MATa ura3-52 MTRJ MATa ura3-52 mtrl-l MATa ura3-52 trpl-Al mtrl-l MATa ura3-52 leu 2-Al lys2-801 ade2-101 mtrl-2 MATa ura3-52 ade2-101 lys2-801 trpl-Al mtrl-3 MATa ura3-52 pTKI-12 MATai ura3-52 ade2-101 lys2-801 his3-A200 trpl-A63 leu2-AJ AMTRJ::LEU2 pTKI -11 MATai mtrl-l cdc28-1 tyri lys2 ade2 his7 adel MATa mtrl-l cdc28-JN ade2 trpl-l his3-11.15 ura3-52 MATa mtrl-l prtl-l tyrl lys2 ade2 his7 ura3-52 AM Ta ura3-52 his3-A200 ACNR1::HIS3 pRSCNRI MATa ura3-52 his3-A200 ACNRI::HIS3 pRSCNR2 MATa ura3-52 his3-A200 ACNR1::HIS3 YEpCNR2 AMTcr ura3-52 leu2-Al ACNR2::LEU2 AMTa/MAMTa ura3-52 his3-A2001his3-A200

YMO107

This study

MATaIMATe ura3-52 leu2-Al/leu2-Al

SP1032 SP6

D.Beach M.Snider

h- piml-46 h- leul-32

prp20-2A 187 185-3-4

YTK102 YTK106 YTK107 YTK108 YTK109 YTK1 10 YTKI 11 YTK1 12 YTK1 13

CNRIIACNRI::HIS3 ACNR2::LEU21ACNR2::LEU2 S.pombe

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T.Kadowaki et al.

abundance, is higher in CNRJ than in CNR2 (0.85 compared with 0.59). We therefore suggest that the putative GTPases which we have identified (CNRI and CNR2 products), as well as Ran and spil mediate the effects of Mtrl/Rccl/piml on RNA processing in all three species.

Materials and methods Yeast strains and DNA manipulation Yeast strains used are given in Table II. The original mtrl-1, mtrl-2 and mtrl-3 mutants were repeatedly backcrossed with YPH45 or 47 (Sikorski and Hieter, 1989). The MTRJ gene was cloned by complementation of YTK102 (mtrl-l) with a yeast YCp5O genomic DNA library (Rose and Broach, 1991). The mtrl-2 and srml-l alleles were rescued by introduction of BamHI-gapped pTK1-9 carrying the MTR1 Satl-XbaI DNA fragment on pRS316 (Sikorski and Hieter, 1989) into YTK107 and SY1115. The mtrl-1 and mtrl-3 alleles were rescued by introduction of MlI-PstI-gapped pTKl-10 carrying the MTR1 Bgml-Sall DNA fragment on pRS314 (Sikorski and Hieter, 1989) into YTK106 and YTK108. Mutations were identified by sequencing the original gapped regions of the plasmids described above with Taq DNA polymerase. For mtrl-l the second position of codon 338 (G) was found to be A, for mtrl-2 the first position of codon 109 (G) was found to be A, for mtrl-3 the second position of codon 464 (G) was found to be A, and for srml-J the second position of codon 117 (G) was found to be A, showing that all are transition mutations. In order to place expression of MTR1 under control of a GAL] promoter, plasmids pTK1-11 and pTKI-12 contain the MTRJ ORF DNA fragment made by PCR with KpnI-digested pTK1-9 as the template and the primers GGAGATCTAATGGTCAAAAGAACAGTCG and GGAGATCTCTTAATCATCCATITCATCC. After Bgll digestion, the PCR product was cloned into the BamHI site between the GAL] promoter and PGK terminator on pRS314 (pTK1-11) and YEp357 (pTKI-12) (Myers et al., 1986). For disruption of the MTRJ gene, pTK1-13 was constructed as follows: the MTRJ BgllI-SalI DNA fragment was cloned into pBluescriptIISK+ and then the internal BamHI fragment (extending from -40 to +798) was replaced by a LEU2 BgmI-Bgm1I DNA fragment. YTK1 11 was constructed as follows: one of the MTR] genes was disrupted by one-step gene replacement using XbaI digested pTK1-13 in a diploid strain YPH501 (Sikorski and Hieter, 1989). Gene replacement was confirmed by Southern hybridization, using the PstI-XhoI MTRJ fragment to probe genomic DNA digested with PstI and XhoI. The strain was then transformed with pTK1-9, followed by sporulation and tetrad dissection. A leu+ ura+ spore was then transformed with pTKl-11 and a resulting leu+ ura+ trp+ clone was inoculated on to a 5-FOA plate containing 2% raffinose and 0.1 % galactose. (High levels of induction were toxic.) A 5-FOA-resistant colony was picked up and grown in SC-LEU, TRP medium containing 2% raffinose and 0.0075% galactose. YTKll1, 112 and 113 were constructed by crossing two parental temperature-sensitive mutants (cdc28-1, cdc28-&N, prtl-l crossed to mtrl-1) with appropriate genetic markers. After sporulation, tetrads containing two ts and two Ts+ spores were selected. The ts spores were backcrossed to parental strains to verify their genotypes. S.pombe strains were grown in YE medium (Matsumoto and Beach, 1991). To detect Ran/Spil homologs, yeast genomic DNA from YPH500 (Sikorski and Hieter, 1989) was used in a PCR reaction with primers corresponding to amino acids 60-67 and 100- 117 in Spilp: 5'-GCA AGC TTT/C AAT/C GTN TGG GAT/C ACN GCN GG-3' and 5'-GCG TCG AC A/G/T ATN GGA/GIT ATA/G TTC/T TCA/G CAN AC-3'. Amplified fragments were gel-purified, digested by HindHI and Sail, and cloned into the corresponding sites of pBluescriptIISK+. For cloning CNR genes, YCp5O yeast genomic libraries (Rose et al., 1987) were screened with the cloned PCR fragments as probes by colony hybridization (Sambrook et al., 1989). DNA sequencing was performed with a Promega TaqTrackTMi Sequencing System. For disruption of CNRJ, the ScaI-PvuH HIS3 gene fragment from pRS303 (Sikorski and Hieter, 1989) was cloned into the EcoRI site of pBTEXCNRlH1.8 (see legend of Figure 4), and the EcoRV-XhoI fragment of the product was used to transform YPH500 and YPH501. Analysis of a genomic Southern blot identified transformants that had one wild-type copy of CNRI and one disrupted copy. For disruption of CNR2, pBTCNR2HB1.2 was digested with TthlllI, and the majority of the coding region (+50 to +610) was deleted with exonuclease III and replaced by a 3.1 kb LEU2 BglJ fragment from YEpl3. The Hindml-BamHI fragment was used for disruption in YPH500 by a one-step gene replacement. After

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recovery of leu+ colonies, analysis of a genomic Southern blot probed with CNR2 sequences confirmed disruption of CNR2. In order to place CNRJ under control of a GALl promoter, the entire CNRl coding sequence was PCR-amplified with the following primers: 5'-GGGGA TCCATGTCTGCCCCAGCTGCTAAC-3'and 5'-GGAAGCTTATAAATCAGCATCATCTTC-3'. After BamHI and HindIm digestion, the PCR fragment was cloned between a GAL1 promoter and PGKterminator on pRS315 (Sikorski and Hieter, 1989) to make pGPCNR1. The strain YMO1 10, in which the CNRI gene is under GAL] control, was constructed by transforming YMO1I1 with the plasmid pGPCNR1 and then curing pRSCNR1 from the transformant by 5-FOA (Sikorski and Boeke, 1991).

Construction of high and low copy plasmids for expression of CNR1 and CNR2. A 1.8 kb HindU fragment containing the CNRI gene was cloned into the HindI site of pBluescriptIISK+, whose EcoRI site had been disrupted, to yield pBTEXCNRlH1.8. A 1.3 kb HindlI -EcoRI fragment containing part of the CNRI gene and a 2.8 kb EcoRI-BamHI fragment containing the rest of the CNRJ gene were cloned into the HindHI -BamHI sites of pBluescriptIISK+ to yield pBTCNRIHB4.1. A 1.2 kb HindmI -BamHI fragment containing the entire CNR2 gene was cloned into the same sites of pBluescriptIISK+ to yield pBTCNR2HB1.2. A BamHI-SalI CNRI fragment from pBTEXCNR1H1.8 was cloned into the same sites of YEp24 to make YEpCNR1. A BamHI-XhoI CNRI fragment from pBTCNRlHB4.1 was cloned into the BamHI-SalI sites of pRS316 (Sikorski and Hieter, 1989) or YEp24 to make pRSCNR1 or YEpCNR14. 1, respectively. A BamHI-SalI CNR2 fragment from pBTCNR2HB1.2 was cloned into the same sites of pRS316 or YEp24 to make pRSCNR2 or YHEpCNR2, respectively. Hydroxyurea treatment Yeast strains were treated with 0.2 M hydroxyurea (HU) in YEPD medium at 25°C (4 h, until they were synchronized with large buds). The temperature was then shifted to 37°C in the presence of HU for 6 h. Serially diluted samples were then inoculated to YEPD plates (lacking HU) at 25°C for several days and the number of colonies was counted.

[3H]methylmethionine and [3H]uridine labeling of rRNA and tRNA YTK100 and YTK102 were grown in SD + URA medium at 25°C then an equal volume of 48°C SD + URA medium was added to yield a 3 ml culture of each. The incubation was continued at 37°C for 30 min, followed by addition of 60 itCi/ml of [3H]methylmethionine (NEN, 72.2 Ci/mmol). After 3 min, 1 ml of cells was poured on to crushed ice in an Eppendorf tube and then centrifuged at 4°C. The rest of the culture was adjusted to 0.5 mg/ml methionine and 1 ml samples of cells were collected as above after 5 and 10 min of continued incubation at 37°C. The extracted RNA was electrophoresed on a 1 % formaldehyde -agarose gel and blotted to Genescreen membrane. The labeled RNA was detected by fluorography using Enhance. EElb (mal-]), pRS316-transformed YTK100 and YTK102 were grown in SC-URA medium at 25°C. An equal volume of 48°C SC-URA medium was added and the incubation was continued at 37°C for 30 min, followed by labeling with 0.5 mCi/ml of [3H]uridine (ICN, 38 Ci/mmol). After 10 min, cells were collected as above and total RNA was extracted. The labeled RNA was electrophoresed on a 10% polyacrylamide-8 M urea gel and then processed for fluorography using Enhance.

Studies of BHK-21 and tsBN2 calls For synchronization, tsBN2 or BHK-21 cells were inoculated at 104 cells/well (24 well dish), cultured 24 hat 33.5 OC in 0.25% serum in DMEM and then shifted to leucine-free DMEM-5% dialyzed serum for 30 h. They were then shifted to 10% serum-DMEM-2.5 mM hydroxyurea for 24 hat 33.5°C and finally transferred to 39.5°C in 10% serum-DMEM2.5 mM hydroxyurea. For the microinjection experiments, cell lines were maintained in Dulbecco's Modified Eagle Medium supplemented with 3.5 g/l glucose, 2mM glutamine and antibiotic/antimycotic mix (Gibco 600-5240AG) 1:100, in a 33°C incubator with a 5% CO2 atmosphere. Cells were laid down at low density on glass coverslips and cultured in the above medium for 4-5 days to promote good attachment to the substrate. The injectate used was p(Ela)BSA: bovine serum albumin with an average of 17.5 residues of Ela peptide (CGYGKRPRPGG) conjugated to it (Breeuwer and Goldfarb, 1990). The conjugate has been labeled with fluorescein (Fl) (as fluorescein-5-isothiocyanate) to -2 mol Fl/mol BSA. Cells on coverslips were incubated at 39.5 C for 6 h prior to injection. They were then rinsed once with PBS and placed into preheated injection dishes containing Hanks' Balanced Salt Solution. The p(Ela)BSA was injected at a concentration of

Yeast RNA transport and GTPases -70 AM in injection buffer (7.6 g/l KCl, 0.64 g/l NaCl, 1.28 g/l K2HPO4, 0.65 g/l KH2PO4, pH 7.0), and the cells were returned to 39.5°C for either S or 30 min. At the end of this time, cells were fixed for 10 min in PBS + 4% formaldehyde and mounted on slides for fluorescence microscopy. Control cells were treated similarly, except that they were removed from the 33°C incubator, injected at room temperature and then returned to the 33°C incubator for 5 or 30 min after injection. They were then fixed for viewing as described above.

Acknowledaements We thank G.Sprague for the srml-l yeast strain, A.Hopper for the rnal-I strain, M.Clark for the prp2O-1 strain, B.Futcher for the cdc28-1N strain, D.Beach for the piml-46 strain, C.O'Dee for the SP6 strain, C.Basilico for tsBN2 cells, R.Sikorski and P.Hieter for plasmids and yeast strains, S.Sazer for allowing citation of unpublished data, D.Templeton for his comments, John Krestnansky for peptides, S.Chen for technical assistance, M.Ward for preparing the manuscript, and NIH grant Nos GM45669 to A.T. and GM40362 to D.G.

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Seino,H., Hisamoto,N., Uzawa,W., Sekiguchi,T. and Nishimoto,T. (1992) J. Cell Sci., 102, 393-400. Sikorski,R.S. and Boeke,J.D. (1991) Methods Enzymol., 194, 302-318. Sikorski,R. and Hieter,P. (1989) Genetics, 122, 19-27. Surana,U., Robitsch,H., Price,C., Schuster,T., Fitch,I., Futcher,A.B. and Nasmyth,K. (1991) Cell, 65, 145-161. Traglia,H.M., Atkinson,N.S. and Hopper,A.K. (1989) Mol. Cell. Biol., 9, 2989-2999. Uchida,S., Sekiguchi,T., Nishitani,H., Miyauchi,K., Ohtsubo,M. and Nishimoto,T. (1990) Mol. Cell. Biol., 10, 577-584. Received on February 23, 1993; revised on April 8, 1993

Note added in proof We have recently obtained direct evidence that CNRJ regulates export of poly(A)+ RNA. A haploid strain with disrupted CNRI and CNR2 genes can be sustained by a centromeric copy of CNR under GALl control, when grown in 0.5% galactose. Upon addition of glucose, growth slows and a nuclear accumulation of poly(A)+ RNA becomes obvious in the majority of cells after 12 - 15 h at 23 'C. The cloning and characterization of CNRl and CNR2 has recently been reported by P.Belhumeur et al. [(1993) Mol. Cell. Biol., 13, 2152-2161]. The EMBL Data Library accession number for the CNRI and CNR2 sequence data are X71945 and X71946, respectively.

Ohtsubo,M., Yoshida,T., Seino,H., Nishitani,H., Clark,K.L. Sprague,G.F.,Jr, Frasch,M. and Nishimoto,T. (1991) EMBO J., 10, 1265-1273. Piper,P. and Aamand,J. (1989) J. Mol. Biol., 208, 679-700. Rao,P.N., Johnson,R.T. and Sperling,K. (eds) (1982) Premature Chromosome Condensation. Application in Basic, Clinical, and Mutation Research. Academic Press. Ren,M., Drivas,G., D'Eustachio,P. and Rush,M.G. (1993) J. Cell Biol., 120, 313-323. Rose,M.D. and Broach,J.R. (1991) Methods Enzymol., 194, 195-230. Rose,M.D., Novick,P., Thomas,J.H., Botstein,D. and Fink,G.R. (1987) Gene, 60, 237-243. Sachs,A. (1990) Curr. Opinion Cell Biol., 2, 1092-1098. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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