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YEAST VOL. 12: 1393-1405 (1996)
Mutational Analvsis of the Gene for Schizosucchavom5ces pombe RNase MRP RNA, mrpl, Using I Plasmid Shuffle by Counterselection on Canavanine JANET L. PALUH? AND DAVID A. CLAYTON*
*Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 943055427, U.S. A. ?Department of Cell and Molecular Biology, 341 Lijk Sciences Addition, University of California, Berkeley, CA 94720-3200, U.S. A.
Received 28 March 1996; accepted 22 May 1996
Reverse genetics in fission yeast is hindered by the lack of a versatile established plasmid shuffle system. In order to screen efficiently and accurately through plasmid-borne mutations in the essential gene for the RNA component of RNase MRP, mrpl, we have developed a system for plasmid shuffling in fission yeast using counterselection on canavanine. The system takes advantage of the ability of the Succhuromyces cerevisiae CAN1 gene to complement a Schizosaccharomyces pombe cunl-1 mutation. Two general use plasmids were constructed that allow directional cloning and initial selection for histidine before counterselection by canavanine. The strain constructed for plasmid shuffling carries auxotrophic markers for ade6, l e d , ura4 and his3 along with the canl-1 mutation. Using this system we examined several partial deletions and point mutations in conserved nucleotides of Schizosaccharornyces pombe RNase MRP RNA for their ability to complement a chromosomal deletion of the mrpl gene. The degree of background canavanine resistance as well as plasmid-plasmid recombination encountered in these experiments was sufficiently low to suggest that the system we have set up for counterselection by canavanine in fission yeast using multicopy plasmids will be widely useful.
KEY WORDS - Plasmid shuffle; RNase MRP RNA; Schizosaccharomyces pombe
INTRODUCTION An important genetic technique available in bud- ding yeast is the ability to analyse a large number of mutations in a particular gene by plasmid shuffle (Sikorski and Boeke, 1991). This method has proved of great value following initial charac- terization of a gene by disruption, and subsequent complementation of the defect by a plasmid-borne copy of the gene. In its most direct use for structure-function studies, this method allows the rapid determination of the phenotypes generated by plasmid-borne mutations in a gene. The effect of the mutation is first measured in the presence of the second plasmid carrying the wild-type copy of the gene. The full extent of the effect of the mutation is then determined upon counterselection *Corresponding Author. tPresent Address.
for cells that have lost the second plasmid and now have no copy of the wild-type gene present. Plasmid shuffle can also be applied to examine synthetic lethal interactions.
The value of a plasmid shuffle system in fission yeast as a tool to analyse plasmid-borne mutations becomes readily apparent when segregation of plasmids through meiosis is examined. The large size of centromeric domains in Schizosucchuro- myces pombe (Carbon and Clarke, 1990) has made the development of centromeric plasmids for use in fission yeast difficult. For plasmids containing an S. pombe arsl element, plasmid stability varies, but is typically 42% in mitosis (Heyer etal., 1986). The ability of the same plasmids to segregate efficiently through meiosis is extremely reduced and often very few spores receive plasmids (Heyer et al., 1986). This segregation deficiency in meiosis makes
CCC 0749-503X/96/141393-13 0 1996 by John Wiley & Sons Ltd
1394 J. L. PALUH AND D. A. CLAYTON
it tedious to analyse a large number of plasmid- borne mutations in a specific gene by tetrad dissection and random spore analysis.
In fission yeast, 5-fluoroorotic acid (5-FOA) has typically been used to select for loss of plasmids carrying the gene for S. pombe ura4+ or Saccharo- m jws cerevisiae URA3 (Grimm et al., 1988; Boeke et al., 1984). The use of 5-FOA has been limited, however, since few auxotrophic markers were generally available for fission yeast and many plasmids and libraries initially constructed often relied on only the S. pomhe uru4+ (Grimm et al., 1988) or S. cerevisiue LEU2 (Beach and Nurse, 198 1) auxotrophic markers. Although more markers have become available, including his7 (Apolinario et ul., 1993), his3 (Burke and Gould, 1994). his1 and his5 (Erickson and Hannig, 1995), and arg3 (Waddell and Jenkins, 1995), the varia- bility encountered in the conditions necessary for stringent 5-FOA selection continues to limit the utility of this approach.
The first report of a plasmid shuffle system in fission yeast (Dandekar and Tollervey, 1992) relied on natural plasmid loss during mitotic growth and used S. cerevisiae 2 p-based plasmids. This method is somewhat tedious, requiring rounds of growth in liquid culture and on plates before replica plating to identify cells containing only the appropriate genetic markers. Recently, Ekwall and Ruusala (1991) reported the ability of the S. cerevisiae CANl gene to complement canl-1 mutation in S. ponzhe. Thus the potential of canavanine as a counterselectable marker in fission yeast has been demonstrated; however, the practicality of using canavanine to select for plasmid loss in a multicopy system has not been investigated.
We describe here the establishment and testing of such a system, including two general use plas- mids containing directional multicloning sites (MCSs), the S. poinbe arsl element, the selectable marker his3, and the S. cerevisiae CANl gene. We have tested this system by analysing mutations in the gene for the RNA component of S. pombe RNase MRP, mrpl. RNase MRP RNA is present in two cellular organelles in vertebrates, suggesting at least a dual cellular function for this ribonucleo- protein (RNP). The RNA has been shown to be present in the mitochondria of cardiomyocytes (Li et al., 1994) and RNase MRP RNP activity isolated from several organisms cleaves in vitro- generated RNA substrates corresponding to the leading-strand origin of mitochondrial DNA rep- lication (Chang and Clayton, 1987a,b; Topper and
Clayton, 1990; Bennett et al., 1992; Dairaghi and Clayton, 1993; Schmitt and Clayton, 1992; Paluh and Clayton, 1995). The majority of RNase MRP RNA is located in the nucleolus (Reimer et al., 1988; Gold et al., 1989) and mutations in the RNA result in a disruption of normal 5.8 S rRNA processing in both budding (Schmitt and Clayton, 1993; Chu et al., 1994) and fission yeast (Paluh and Clayton, manuscript in preparation). The gene for this RNA has been shown to be essential in both S. cerevisiae (Schmitt and Clayton, 1992) and in S. pombe (Paluh and Clayton, 1995) although the nature of the essential phenotype is unknown.
While the degree of homology between human and yeast MRP RNAs is less than 50 percent, RNase MRP RNAs display considerable conser- vation from human to yeast in the predicted struc- ture of the RNAs (Schmitt et al., 1993). S. pombe mrpl was cloned by homology to metazoan RNase MRP RNAs (Paluh and Clayton, 1995), and simi- larities between S. pombe and human cells in both mitochondrial genome size and organization of mitochondrially encoded genes (Paluh and Clayton, 1995) make S. pombe an ideal system to continue studies of mitochondrial functions of RNase MRP in relation to metazoan RNase MRP RNAs. In order to facilitate mutational analysis of conserved domains and nucleotides of the RNA, we have established a plasmid shuffle system for use in fission yeast.
MATERIALS AND METHODS
Yeast strains, media and genetic methods S. pombe strains are listed in Table 3 . The strains
FY255 and FY392 were a generous gift from Dr Susan Forsburg (Salk Institute, San Diego, CA). Standard genetic procedures, lithium acetate trans- formation and EMM media were as described (Moreno et al., 1991). Strains were grown on either MSA (Egel et al., 1994) or EMM minimal media supplemented with the appropriate amino acids (75 ,ug/ml histidine, uracil and leucine and 100 pg/ ml adenine) and for counterselection contained 60 to 75 pg/ml canavanine (Sigma, S Louis, MO).
Construction ojplasmids f o r counterselection The plasmids pBGl (Burke and Gould, 1994)
and pUC19-CAN1 were kindly provided by Dr Susan Forsburg (Salk Instifute, San Diego, CA). A KpnI-Pstl fragment containing the CAN1 gene from pUCl9-CAN was cloned into the same
PLASMID SHUFFLE IN S. POMBE
Table 1.
1395
Oligonucleotides used for PCR site-directed mutagenesis.
KOC- 1 KOC-2
ST54A ST54C ST54B
3ENDA 3ENDB
LOOPA 1 LOOPA2
LOOPB 1 LOOPB2
STEM 1A STEMlB
STEM2A STEM2B
STEM6A STEM6B
LRBl LRB2
TOptA TOptB
DEGlA DECIB
S’-TGCTATGTCGGTCAATTGCTCGTCG-3’ 5’-CGCTATCGCGTTTCAGTGCTTGAC-3’
S’-TGACTGCTTCAACCACTCGTTCGAGCTCAAAGG-3‘ S’-TGGTTGAAGCAGTCATACAGG-3’ 5’-TCACTGCTTCAACCACGATCGGAGCTCAAAGGTATC-3’
S’-GGGGCTTAGTCTCAAAGGTTTTTTTCC-3’ 5’-GGAAAAAAACCTTTGAGACTAAGCCCC-3’
S’-AAGCCCCCTTAGTCTTACATCAACAAATGG-3‘ 5’-TTTGTTGATGTAACACTAAGGGGGCTTAG-3’
S’-CGTCGTAGTTGAACGTGATACGGTCGCATCC-3’ 5’-GCGACCGTATCACGTTCAACTACGACG-3’
S’-CCCCGGACTCTTGCGTCAACCTCTCCATGG-3’ S’-CCATGGAGACGTTGACGCAAGAGTCCGGGG-3’
S’-CGGTAACCTCCTCAATTAAAGAATCAGCGG-3’ S’-CCGCTGATTCTTTAATTCAGCACGTTACCG-3’
5’-GAAGAATCAGCGGTAATGGCGTAGTTTTTC-3’ S 'GAAAAACTACGCCATTACCGCTGATTCTTC-3'
S’-CAGCTTTAGGGTTTGTCCCCGGACTC-3’ 5’-CCGGGGACAAACCCTAAAGCTGTTTAG-3’
5‘-GATCGTGGTTCAAAAAGTCATACAGGAA-3‘ 5’-TTCCTGTATGACTTTTTCAACCACGATC- 3’
5’-TGATAGTTAT(AG)CG(GA)T(CT)GCAT(AC)CA(TA)(TA)TGTTGATG-3’ 5‘-ATAACTATCAACTACGACGAAAA-3‘
unique sites in pBGl to generate pJPpsl. The KpnI site and an adjacent Sac1 site were removed from pJPpsl by digestion and filled in with Klenow to produce pJPpsD. Two oligonucleotides, psA,S’-
TCACCATGGATAGGTACCAACTGCA-3’ and
TGCGGCCGCTGAGCTCCAGATCTATGCA- 3‘, were annealed and cloned into the PstI site of pJPpsD to generate the directional MCSs in pJPps3 and pJPps4.
Construction of mrpl mutations Plasmid pURN18 (Barbet et al., 1992) was a
kind gift from Dr Tony Carr (MRC, University of Sussex, U.K.). All mrpl mutations were cloned into pURN18 on a Pstl-XbaI 1.2 kbp fragment containing the native mrpl promoter. Convenient
TAGATCTGGAGCTCA-GCGGCCGCAGATA
PSB, 5’-GTTGGTACCTATCCATGGTGATATC
restriction sites in the S. pomhe mrpl RNA gene (Paluh and Clayton, 1995) were used to generate deletions mrpl-ND90 (NcoI to DraI, deletion of nucleotides 111 to 198) and mrpl-RRYO (RsaI to RsaI, deletion of nucleotides 199 to 282) in pURN18. Mutation mrpl-TOd removes nucleotides 34 to 74 and was generated by site- directed deletion mutagenesis (Kunkel, 198.5; Eghtedarzadeh and Henikoff, 1986) using oligo- nucleotide TOd- 1, S-TCGAACGATCGTGG-
Oligonucleotides used for two step polymerase chain reaction (PCR) generation of mutations are listed in Table 1. Oligonucleotides KOC-1 and KOC-2 are complementary to flanking sequences just outside of the sequence encoding the mature RNase MRP RNA. KOC-1 and KOC-2 were first used with the appropriate internal oligonucleotides
TTAGGGAAAGTCCCCG-3’.
1396 J. L. PALUH AND D. A. CLAYTON
0 Point mutation @ Point insertion U Large deletion 17 Small deletion
Cage region
A A U G A
AG A-U U-G
A A C-G
A-ti 250--OU_lp
C-G U-A
A-U U-G G-U A-U
C
uG-C ,,GU c
Guuuc u-; U-A
270 Gc A
Q-u U-A C-G-210
cti G U G ~ U - A G A A
--"AcG AAC' A-U '- A-U
Q-u GC-195 U-A A-U
C
U-G U
U u
D C
Figure 1. Mutational analysis of the gene for S. pornbr RNase MRP RNA. The sequence of the RNA and its predicted secondary structure are shown. Mutations are described in detail in Table 4 and Materials and Methods. Numbering is indicated along with the sequence. The curved line between nucleotides 77 and 78 represents continuity in the sequence and a three-dimensional folding of the structure. The LRI in the cage region is indicated by four vertical bars between nucleotides 84-87 and 377-381.
(shown in pairs) to generate PCR products consist- ing of partial fragments of mrpl and flanking sequence containing the site-directed mutation. Mutations mrpl -ST54A and mrpl -ST54B used the same oligonucleotide, ST54C, for mutagenesis. These first-round PCR products were then mixed and extended with KOC-1 and KOC-2 to generate a PCR fragment containing the entire mrpl gene and flanking sequence. Conditions for PCR were essentially 94°C 1 min, ice 1 min, 72°C 1 min (five rounds); 94°C 1 min, 35°C to 45°C 1 min, 72°C 1 min (30 rounds); 72°C 5 min (one round). PCR- generated mutations in mrpl were first cloned
into pPCRII (Invitrogen, San Diego, CA) and confirmed by Sanger dideoxy sequencing then recloned into pURNl8. Oligonucleotides for sequencing mrpl have been described (Paluh and Clayton, 1995).
RESULTS Generation ojmutations in mrpl and genetic analysis of mutations mrp1-ND90 and mrpl-RR90 as controls j o r plasmid shufle
To provide insights into the nuclear and mitochondria1 roles for RNase MRP, we have
PLASMID SHUFFLE IN S. POMBE
mutagenized the gene for the RNA component of S. pombe RNase MRP, mrpl. Locations of the mutations constructed in mrpl are shown in Figure 1. Details of the mutagenesis are given in Materials and Methods and the oligonucleotides used for site-directed PCR mutagenesis are listed (Table 1). Sites to be modified were chosen for their conser- vation in heterologous RNase MRP RNAs and for their similarities to or distinctness from conserved nucleotides present in the cage region (Figure 1) of the related RNase P RNAs (Forster and Altman, 1990). Each plasmid-borne mrpl mutation is present on an identical fragment as that for wild- type mrpl and utilizes the native mrpl promoter.
Two mutations in m r p l , mrpl-ND90 and nzrpl- RR90, were chosen for initial characterization by standard genetic methods and were used as controls in the plasmid shuffle system. The plas- mids carrying these mutations, pJPurND90 and pJPurRR90, were transformed into the diploid JLP99 (Paluh and Clayton, 1995) followed by tetrad dissection and random spore analysis. No spores that were uru+ (plasmid marker), leu+(mrpl::LEUZ) were found for plasmid pJPurND90; however, pJPurrRR90 was able to complement (Table 3, strain JLP106; Table 4). The growth rate of JLP106 is slightly reduced compared to JLPlOO (data not shown).
1397
chromosomal knockout of the mrpl gene (mrpl::LEU2) was transferred to the genetic back- ground containing canl-1 and his3-Dl by crossing strain JLP201 (h+) with JLPlOO (h90), followed by random spore analysis and selection for uru + , leu+ isolates. From this cross, strain JLP204 (h+) was isolated and transformed with pJPps2b to generate JLP205 which contains two plasmids car- rying identical fragments of the wild-type mrpl gene and either the gene for ura4 (pJPurWT) or the genes for both his3 and CAN1 (pJPps2b). JLP206, which contains only pJPpBb, was generated by streaking JLP204 cells onto medium containing 5-FOA and testing resistant clones for his+ prototrophy and ura auxotrophy.
Final strains for plasmid shuffle were tested for expected resistance or sensitivity to canavanine (Figure 3A). As expected, strains JLP201 and JLP204 (carrying mrpl on pJPurWT) are able to grow on canavanine since no wild-type copy of the CAN1 gene is present. JLP205, which carries two plasmids with the mrpl gene (pJPurWT and pJPps2b) is able to survive after loss of pJPps2b carrying the CAN1 gene. JLP206 cells do not grow on canavanine since both loss of pJPps2b, carrying the essential gene mrpl , and retention of the CAN1 gene on this plasmid are lethal consequences.
Strains and plasmids jbr a canavanine-based plasmid shufle system
The plasmids used for counterselection are shown in Figure 2A along with a diagram of the plasmid shuffle scheme in Figure 2B. The original plasmid, pJPps1, was modified as described in Materials and Methods to generate two general use plasmids, pJPps3 and pJPps4, that have oppositely oriented MCSs to allow for directional cloning (Table 2). pJPurWT has been described (Paluh and Clayton, 1995) and is able to comp- lement a chromosomal deletion of mrpl (Table 3 , strain JLPlOO and JLPlOl). pJPps2b and either pJPurWT (as a positive control) or mutations in mrpl in pURN18 (Table 4) were used for plasmid shuffle.
To generate the appropriate general strain for plasmid shuffle, the his3-DI and canl-1 mutations were combined. Table 3 lists all strains used in this study. Strain FY255 (h+), containing the c a d - 1 mutation, was crossed with strain FY392 (h - ), carrying the his3-Dl marker, to generate strain JLP201 (h+). For plasmid shuffle of mrpl, the
Analysis of plasmid-borne mutations in mrp 1 by coun terselection on cana vanine
Plasmids containing mutations in the mrpl gene (Figure 1 and Table 4) were transformed into strain JLP206 and plated on MSA supplemented with adenine and histidine (MSA + AH). Trans- formants that were ura+ were selected and replated onto the same medium before testing for the ability to lose pJPps2b by growth on EMM medium supplemented with adenine, histidine and canavanine (EMM + AHCAN). Several isolates of each transformant were tested against cells shuffled with control plasmids pJPurWT, containing the wild-type copy of mrpl, and pJPurND90, which contains the non-complementing partial deletion of mrpl. In Figure 3B, duplicate samples of those cells carrying plasmids with mrpl mutations that are able to complement after plasmid shuffle are shown streaked on EMM+AHCAN. Figure 3C shows streaks of multiple isolates of cells trans- formed with plasmids containing mutations in mrpl which did not complement. All mrpl mutations examined by this method were readily scored for their ability or inability to complement.
1398
A. J. L. PALUH AND D. A. CLAYTON
sad Kpnl
Figure 2A.
Relatively little background was observed and EMM+ AHCAN plates. Plates were placed at always occurred in the most dense part of the 30°C, 36°C and 17°C for 6-20 days to check streak. The results of complementation after for temperature-sensitive or cold-sensitive muta- plasmid shuffling are summarized in Table 4. tions (Figure 4.) The growth rate of mrpl-RRYO
After testing multiple isolates at 30”C, a single containing cells on plates is noticeably slower than representative isolate for each mutation was that of the same strain carrying the plasmid with streaked in triplicate from MSA+AH plates to the wild-type copy of i?zrpI and is exacerbated
PLASMID SHUFFLE IN S. POMBE 1399
WTmrpl mrpl mutation to be tested
Before growth on canavanine
Wild-type mrpl present (CANr, his+, urn+)
1 + canavanine
Deletion canl-1 mrpl
ura4+
(complementing) mtpl mutation
0 Following counterselection
( C A P , uru+) Viable if mrpl with mutation
remains functional
Figure 2. Counter selectable plasmids for use in plasmid shuffle. (A) The general use plasmid pJPps3 was derived from pJPpsl. The unique restriction sites present in the MCS of pJPps3 and pJPps4 are shown. pJPps2b, containing wild-type mrpl , was used for plasmid shuffle with either pJPurWT or plasmids containing partial deletions of the mrpl RNA in pURN18 (see Table 4). Details of plasmid construction are described in Materials and Methods. Plasmids are also de- scribed in Table 2. (B) Diagram of the plasmid shuffle scheme used to analyse mutations in mrpl. A bar represents the chromosomal canl-1 gene and deleted mrpl gene. Plasmids are shown by circles in single copy for simplicity, but contain the S. pombe ursl sequence and are multicopy.
when cells carrying this plasmid are grown at 36°C. Both the slower growth rate and temperature sensitivity were evident using the plasmid shuffle system (controls, Figure 4). Cells carrying another mutation, mrpl-ptl-9, also showed this same pattern (streak 11, Figure 4). The slight growth observed with cells carrying the mrpl-3'END mutation at 17°C and 30°C was not reproducible (streak 12, Figure 4).
Spontaneous reversion to canavanine resistance was determined by plating strains JLP201 and JLP206 on EMM with or without canavanine. Recombination between the plasmid bearing the mrpl mutation and wild-type plasmid was assumed to be occurring at a low frequency since some of the colonies in the dense part of the streak were found to be his - , canavanine resistant, ura+. These were considered false positives since their appearance was not reproducible from streaks of multiple isolates and these cells resembled wild- type cells in their growth and appearance.
DISCUSSION S. pombe RNase MRP RNA was cloned by homology to metazoan RNase MRP RNAs (Paluh and Clayton, 1995). In order to define func- tional domains and key nucleotides in the RNA component of RNase MRP from fission yeast, we have mutagenized the gene using site-directed approaches that target conserved features. To facilitate analysis of these plasmid-borne muta- tions in mrpl and as an alternative to available standard genetic approaches, we have established a system of plasmid shuffle by counterselection on canavanine for fission yeast.
Plasmid shufle in fission yeast using counterselection on canavanine
Plasmid shuffle for analysis of mutations in yeast is an established tool in S. cerevisiae. In S. pombe,
Table 2.
Plasmid Relevant Genotype mrpl Fragment Base plasmid
S. pornbe plasmids used in this study.
pJPurWT rnrpl+, ura4+, arsl 1.1 kbpPstl-Xbal pURN18 (Tony Carr) pJPpsl his3 -+ , CA N l , arsl None pBGl-his3+,puclO-CANl (Susan L. Forsburg) pJPps2b his3 +, CANl, rnrpl+, arsl 1.1 kbpPstl-Xbal pURN18, PCRII (Promega) pJPps3 his3 + , CANI, arsl None pJPpsl (this work) pJPps4 his3 +, C A N l , arsl None pJPpsl (this work)
e
P
0
0
Tabl
e 3.
S.
pom
be s
train
s us
ed in
this
stu
dy
Stra
in
Gen
otyp
e So
urce
JLP9
9 h+
/h+
ad
e6-M
210l
ade6
-M21
6 le
ul-3
2lle
ul-3
2 ur
u4-D
18/u
ra4-
D18
mrp
l +lm
rpl:
:LE
U2
Palu
h an
d C
layt
on (
1995
) JL
PlO
O
h90
ade6
-M21
0 le
ul-3
2 ur
a4-D
l8 m
rpl:
:LE
U2[
pJP
urW
T,m
rpl+
, ura
4+]
Thi
s w
ork
JLPl
Ol
h+
ade6
-M21
0 le
ul-3
2 ur
a4-D
18m
rpl:
:LEU
2[pJ
PurW
T, m
rpl +
, ura
4+]
Thi
s w
ork
JLPl
O4
h + Ih
+ ad
e6-M
2101
ude6
-M21
6 le
ul -3
2/le
ul-3
2ura
4-D
18/u
ra4-
D18
mrp
l + lm
rpl:
: LE
U2[
pJPu
rND
90,
Thi
s w
ork
JLPl
O6
h90
ade6
-M21
0 le
ul-3
2 ura4-D18mrpl::LEU2[pJPurRR90, m
rpl-
RR
90, u
ra4 +
] T
his
wor
k m
rpl-
ND
90,u
ra4+
]
FY25
5 h+
ad
e6-M
210
led-
32 u
ra4-
Dl8
can
l-1
Susa
n L
. For
sbur
g FY
392
h -
ade6
-M2 1
0 le
ul -3
2 ur
a4- D
l8 h
is3-
DI
Susa
n L.
For
sbur
g JL
P20 1
h
+ ad
e6-M
210
leul
-32
ura4
-DI8
his
3-D
l ca
nl-1
JL
P204
h +
ad
e6-M
210
leul
-32
ura4
-Dl8
his
3-D
I ca
nl-l
mrp
l::L
EU
2[pJ
Pur
WT,
mrp
l+, u
ra4+
] T
his
wor
k JL
P205
h+
ad
e6-M
210
leul
-32
ura4
-DI8
his
3-D
l ca
nl-l
mrp
l::L
EU
2[pJ
Pur
W~,
mrp
l+, u
ra4+
; pJP
ps2b
, T
his
wor
k
JLP2
06
h +
ade6
-M21
0 le
ul-3
2 ur
a4-D
l8 h
is3-
DI canl-lmrpl::LEU2[pJPps2b, m
rpl+
, CA
NI,
his
3+]
Thi
s w
ork
mrp
l + , C
A N
l , hi
s3 + ]
c T
PLASMID SHUFFLE IN S. POMBE
Table 4.
1401
Plasmid shuffle phenotypes of mrpl mutations
Mutation" mrpl Fragment Plasmid shuffle
Phenotype"
ST54A ST54B 3'END LoopA mLoopB Stem1 Stem2 Stem6 LRB TOpt TOdel Ptl-2 Ptl-3 Ptl-9 ND90 RR90
(Del 26 to 27) -
(Del 20 to 22) (Del 391 to 396) -
(U+A (371), C+A (372), Ins G (374-375), U-G(377)) ~
(U-A (339), A-C (340) U - + G (343)) __
(C-A (141), G-A (144)) ~
(A-U (79), A-U (SO), A-U (81)) ~
(G+A (37), C+A (38)) -
(Del 34 to 74) -
~
(U+A (101), U+A (102), G+A (103), A-C (104))
(Del 157 to 162) + + + + + + + +
(U+A (360)) + + + + (U-A (359)) + + + + (U-A (359), C+A (356))
(Del 199 to 282)
+ + + (ts)
+ + + (ts) (Del 111 to 198) ~
"The base plasmid is pURN18 (ura4+, ars f ) . Plasmids carrying mutations are referred to as pJPur derivatives in the text (ex. pJPurST54A). "Non-viable phenotypes after plasmid shuffle are designed by a dash (-). Growth comparable to wild-type is designated as + + + +. Temperature sensitivity (ts).
the lack of such a system has been exacerbated by the poor segregation of S. pombe ars-containing plasmids to daughter cells during meiosis. The system we have set up for characterizing plasmid- borne mutations utilizes the strain JLP201 that carries the auxotrophic markers his3, ade6, ura4 and leu1 along with the mutation cad-1 , pro- viding resistance to canavanine. Two general use plasmids were designed that allow directional cloning and provide initial selection with his3 and then counterselection of the same plasmid, via the S. cerevisiae CAN1 gene, with canavanine. Since S. pombe ura4 and S. cerevisiae LEU2 genes are often used as markers for gene disruption and plasmid rescue, the strain and plasmids designed here for counterselection accommodate such previous use of those markers. The plasmid, pURN18, was used in combination with the plas- mid shuffle vectors designed here and has been described previously (Barbet et al., 1992). No promoter sequences are present in any of the plasmids described, requiring expression of the gene of interest from its own promoter or available regulatable promoters (for review see Forsburg, 1993). Constructs containing mrpl expressed from pREP4 (Maundrell, 1993) modified for RNA ex-
pression (data not shown) also worked well in this system.
Two types of revertants were expected to be present using counterselection on canav- anine: background resistance to canavanine and plasmid-to-plasmid recombination. Both his+, canavanine-resistant clones and ura+, his - , canavanine-sensitive false positives were encoun- tered. Little or no revertants are observed in cases where one plasmid is present or when heterologous RNase MRP RNA sequences are examined (Paluh and Clayton, unpublished observations). The highest amount of suspected plasmid-to- plasmid recombination occurred when both plasmids contained only minor differences in the gene sequence of interest, including the flanking sequence. This suggests that the base plasmid sequences themselves do not represent a significant contribution to background from recombination. The size of the fragment examined in these exper- iments, including both the mrpl gene and its regulatory sequences, is only 1.2 kbp. A higher frequency of plasmid-to-plasmid recombination would be predicted when examining larger Srag- ments containing protein-encoding genes in such a system. Therefore the degree of background
J . L. PALUH AND D. A. CLAYTON
should be determined initially by examining a few mutations, using both standard genetic methods of random spore analysis and plasmid shuffle, and then testing multiple transformants for each plasmid-borne mutation examined.
Mutations in S. pombe mrpl that target identified sites of protein interaction
Few protein components for eukaryotic RNase MRP have been identified. Coimmunoprecipita- tion of both RNase MRP and RNase P RNAs by human autoantisera suggests that there is a shared protein component between RNase MRP and RNase P RNAs (referred to as the To or Th antigen; Reddy et al., 1983; Gold et al., 1989). In S. cerevisiae, the Pop1 protein has also been shown to be a shared protein component of RNase MRP and RNase P RNAs (Lygerou et al., 1994). The mrpl-ToDel mutation removes sequences in rnrpl corresponding to the same region in human RNase MRP RNA known to be important for binding the To antigen in human cells (Yuan et al., 1985). The inability of this mutation to complement is there- fore consistent with what is predicted based on the situation in humans. Both nucleotides altered in the non-complementing mutation rnrpl-TOpt have been changed to match nucleotides present at a similar region of the RNase P RNA. Whether these are critical nucleotides required for rnrpl function or whether the defect is simply due to altered pairing within the stem is not clear.
In S. cerevisiae RNase MRP RNA a single point mutation, at a position similar to the S. pombe mrpl-Stem2 point mutations, was shown to re- sult in a temperature-sensitive lethal phenotype (Schmitt and Clayton, 1994; Chu et al., 1994). The point mutations for mrpl-Stem2 are not tolerated and, unlike the case for a single substitution in S. cerevisiae MRP RNA, no temperature- sensitivity is associated with the dual changes. The mrpl-Stem2 mutation would be expected to have little effect on stability of this stem and loop structure since the altered nucleotides are predicted to be unpaired. It is more likely that these point mutations affect nucleotides involved in long-range interactions within the RNA or represent a site of RNA-protein binding similar to the case in S. cerevisiae.
Rigid requirements for conserved sequences within the cage region of S. pombe RNase M R P R N A
Mutations in mrpl were designed to clarify our
1402
A.
J L P 2 O l
JLP205
JLP206
B* Stem1
Stem 6
C.
JLP204
WT
Pt 1-9
Figure 3. Plasmid shuffle by counterselection on canavanine. (A) Strains constructed for use in plasmid shuffling were streaked to EMM supplemented with adenine. histidine. uracil, leucine and canavanine (EMM +AHULCAN) to verify ex- pected sensitivity or resistance. The strain genotypes are given in Table 3 . (B) Duplicate streaks of those isolates which are able to complement a chromosomal deletion of mrpl following plasmid shuffle. (C) Multiple streaks of those isolates which failed to complement a chromosomal deletion of mrpl by plasmid shuffle are shown, along with pJPurND90 (a, negative control) and pJPurWT (b, positive control). Isolates x1-x3 and yl -y3 represent three random transformants chosen to be analysed for each mutation. The identity of the mutations is shown along with each plate. The plate containing the mutation LRB contains only that mutation for testing along with the described controls. understanding of critical doma& and nucleotides
PLASMID SHUFFLE IN S. POMBE 1403
Controls
i T C
W C
36°C
17c
W C
36'C
1)LoopA 5)Todel 9) STYIB 13) Stem6 2) WT 6) TOpt 10) ST51A 14) Stem2 3) Ptl-2 7) LRB 11) Pti-9 15) Stam1 4) Ptl-3 8) LoopB 12) S'END
Figure 4. Screening for temperature-sensitive mutations. After testing of multiple isolates for each mutation transformed into JLP206, representative isolates were streaked on EMM+AHULCAN plates and incubated at 17°C (20 days), 30°C (6 days) or 36°C (12 days). Fourteen plasmids bearing m p l mutations were analysed along with pJPurWT and ND90 and RR90 controls.
present within the RNA. Several mutations tar- geted conserved nucleotides within the cage region of RNase MRP RNAs (Figure 1). The structure of the cage region of RNase MRP and RNase P RNAs has been predicted by phylogenetic comparison and is conserved between these two RNAs. Mutations within this region that signifi- cantly alter the predicted structure (mrpl -TOdel and mrpl3 'END) resulted in loss of mrpl func- tion. In addition, all mutations that altered conserved nucelotides (mrpl -ST54B, mrpl -Loop B, mrpl-TOpt and mrpl-LRB) or were designed to either introduce pairing (mrpl-LoopA) or reduce pairing (mrpl-ST54A and mrpl-LRB) in proposed stem and loop structures within this region were
deleterious. Interestingly only those mutations that altered sequences within the small stem and loop structure near the centre of the cage region re- mained functional for complementation. While sequences within this stem and loop and at the central core of the cage region of the RNA are nearly identical between S. pombe RNase MRP and S.pombe RNase P RNAs, they are less well conserved between yeast and human RNase MRP RNAs (Paluh and Clayton, 1995; Schmitt et af., 1993).
In general, mutations affecting sequences out- side the cage region were tolerated. Both mrpl- Stem1 and mrpl -Stem6, which were designed to destabilize predicted stem and loop structures in
1404
the RNA, had no affect on viability. Mutation mrpl -RR90, which removes a significant portion of the longest predicted stem and loop structure of the RNA, is also viable. Only deletion mrp-ND90, which removes a large portion of the central sequence of the RNA, and the point mutations for the mrpl-Stem2 mutation were non-viable. It re- mains possible that subtle alterations in rRNA processing or mtDNA replication are associated with some of the viable mrpl mutations. Further analysis of cells carrying these mutations may provide insights into understanding mrpl function in fission yeast.
Since little information is available on the exact nature of the protein components for the RNase MRP RNP and limited sequence identity exists between heterologous RNase MRP RNAs, mutational analysis of the gene encoding the RNA component of this RNP represents the best current approach towards deciphering the essen- tial functions of RNase MRP. The majority of the mutations in mrpl tested by plasmid shuffle in this study were unable to complement a chromosomal deletion of mrpl. However, two temperature-sensitive mutations, mrpl-Ptl-9 and mrpl- R R90, were tested that showed reproduc- ibly poor growth at 36°C versus 30°C (Figure 4 controls and streak 11). Further analysis of cells carrying these mrpl mutations and identification and characterization of additional partially com- plementing mutations in the mrpl gene using the plasmid shuffle system should lead to insights into the nuclear and mitochondrial functions of RNase MRP.
J. L. PALUH AND D. A. CLAYTON
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ACKNOWLEDGEMENTS
We thank Dr Susan L. Forsburg for suggesting counterselection by canavanine and for providing starting plasmids and strains, Dr Teresa S.-F. Wang laboratory members, Martha Arroyo, Dipa Bhaumik, and Dr Ivo Galli for their helpful comments during set up of the plasmid shuffle system and Dr Tim Brown and Jin Shang for their invaluable comments on the manuscript. J.L.P. is supported by grant GM33088-25 from the National Institute of General Medical Sciences. This work was supported in part by Public Health Service grant 5T32CA09302 awarded by the National Cancer Institute, DHHS.
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