Apr 5, 2016 - Needleman and Tzagoloff (30). Proteins were separated on ..... Herbert, C. J., Labouesse, M., Dujardin, G., and Slonimski, P. P. 18. Shraphin, B.
Vol. 267, No. 10, Issue of April 5,pp. 6963-6969, 1992 Printed in U.S. A .
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc
The Nuclear GeneMRSB Is Essential for the Excision of Group I1 Introns from Yeast Mitochondrial Transcripts in Vivo* (Received for publication, June 3, 1991)
Gerlinde WiesenbergerS, Martin Waldherr, and Rudolf J. SchweyenQ From the Institut fiir Mikrobiologie und Genetik, Universitat Wien, A-1090 Vienna, Austria
circular RNAs, the so-called lariats, with a most intriguing RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several 2’-5‘ phosphodiester bond. This relatedness has led to the proteins encoded by nuclear genes. In this study we speculation that the autocatalytic group I1 introns are the report on the isolation, nucleotide sequence, and pos- evolutionary ancestors of the nuclear (non-autocatalytic) sible functions of the nuclear MRS2 gene. When pres- mRNA introns (10). MRSB gene acts ent on high copy number plasmids, the The finding of self-catalysis of mitochondrial introns in as a suppressor of various mitochondrial intron muta-vitro contrasts a number of observations which have revealed tions, suggesting that theMRS2 protein functionsas a that excision of group I and group I1 introns in vivo involves splicing factor. This notionis supported by the obser- proteins (see Refs. 11and 12 for review), encoded either within vations that disruption of single the chromosomal copy the intronsthemselves, the so-called maturases, or by nuclear of theMRSB gene causes(i) a pet- phenotype and(ii) a genes. Two of these nuclear genes in yeast, MRSl andCBP2, block in mitochondrialRNA splicing of all four mito- encode products that areneeded only for the excision of group are effichondrial group I1 introns, some of which ciently self-splicing in uitro. In contrast, thefive group I introns (b13 and aI5b b15, respectively); yeasts lacking the I introns monitoredhere are excised from pre-mRNA introns in question are unaffected by disruption of these in a MRS2-disrupted background although at reduced nuclear genes (13-15). Others encode mitochondrial proteins rates. So far the MRS2 gene product is unique in that of dual function,like CYT18 in Neurospora crassa and NAMZ addition it is essential for splicing of all four group I1 introns, in yeast which serve as tRNA synthetases and are in but relatively unimportant for splicing of group I in- essential splicing factors (16, 17). Finally, the yeast MSS116 trons. In strains devoid of any mitochondrial introns gene encodes a protein with similarity to a helicase; its absence the MRSS gene disruptionstill causes apet- phenotype affects the excision of both group I and group I1 introns and and cytochromedeficiency, although the standard pat-other, so far unknown functions (18). tern of mitochondrial translation products is produced. We previously described the isolation of three nuclear Therefore, apart from RNA splicing, the absence of thegenes, MRS2, MRS3, and MRS4, whose products appear to MRSS protein may disturb the assembly of mitochon- be involvedin theexcision of group I1 introns from mitochondrial membrane complexes. drial pre-mRNA (19).They have been detected by virtue of their ability to suppress a splice defect exerted by a group I1 intron mutation, when present in high copy number. MRS3 and MRS4 were found to be closely related genes; Organelle introns have been classified as group I and group both can be disrupted without causing any phenotypically 11 introns according to conserved secondary structure features detectable effect on splicing or any other function (20, 21). and group specific excision pathways (1,2). Group I1 introns We show here that the MRS2 gene, unlike the MRSB and have been detected so far in mitochondrial and chloroplast MRS4 gene, is essential for the excision of all four group I1 genomes whereas group I introns are widespread in other intronspresent in yeast mitochondrial RNAs, whereas it genomes, namely in the nuclear rRNA genes of Tetrahymena hardly affects the excision of group I introns. MRSB has additional, so far unknown functions since its disruption in and ingenes of bacteriophages and cyanobacteria (1-6). Some members of each group have been shown to be cata- yeast strains lacking all known introns still leads to respiralytic in vitro; they excise themselves from pre-mRNA and tory deficiency. ligate the exons in the absence of any protein (7-9). The MATERIALS AND METHODS splicing pathway and conserved sequence elements at the splice junctionsrelate group I1 introns with the nuclear Strains and Plasmids-The genotypes and origins of the yeast mRNA introns; bothclasses of introns areexcised as branched strains used in this study are described in Table I. Escherichia coli
* This work was supported by the Austrian Fonds zur Forderung der wissenschaftlichen Forschung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s)reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accessionnumbetfs) M82916. $ Present address: Section of Genetics and Development, Cornel1 University, Ithaca, NY 14853-2703. 8 To whom reprint requests should be addressed Institut fiir Mikrobiologie und Genetik, AlthanstraRe 14, A-1090 Wien, Austria. Tel.: 1-31-336-1392:Fax: 1-31-336-700.
strain XL1-Blue (Stratagene) and the following plasmids were used for subcloning: YEp351 (24), Bluescript (Stratagene). Rho” derivatives of yeast strains were obtained by growing cultures for about 30 generations in YPD medium containing 50 pg/ml ethidium bromide. Media-Yeast strains were grown in complete medium YP (1% yeast extract, 2% peptone) enriched with either the fermentable carbon sources glucose(2% (w/v) glucose, YPD), raffinose (2.5% raffinose, YPR) or with the nonfermentable carbon source glycerol (3% w/v glycerol, YPG). YPG was occasionally supplemented with 0.05% glucose (YPdG) to improve initial growth of the cells after replica plating. Synthetic minimal media (0.67% Difco yeast nitrogen base, 2% glucose (SD) or 2.5% Raffinose (SR) were supplemented with amino acids and bases when appropriate.
6963
6964
Control of Group 11 Intron Splicing TABLEI Genotypes and sources of yeast strains used Mitochondrial genomes are indicated in brackets. If not otherwise stated, rho+ strains of S. cereuisiae contain introns bI1, bI2, bI3, bI4, and bI5 of the COB gene, introns aI1, aI2, aI3, aI4, aIBa, aIBb, and aI5c of the COXl gene, and the 21 S rRNA intron w. Ab1 and AaI indicate that the respective introns of COB or COXl are deleted, whereas AxbI and AcaI indicate that all introns are deleted. The mitochondrial genome of Saccharomyces uuarum (SU) contains the following introns: b14, bI5; a12, aI3, aI5c (22). EtdBr, ethidium bromide. Strain
DBY747 DBY7471M1301 DBY747/rhoo
Genotype: nuclear [mitochondrial]
MATa leu2-3 leu2-112 his3-1 u r d - 5 2 trpl-289 [rho+ mit+] MATa leu2-3 leu2-112 his3-1 u r d - 5 2 trpl-289 [rho+mit~M13011 MATa leu2-3 leu2-112 his3-1 u r d - 5 2 trpl-289
frh.0~1
GW7/gd2-21.2
MATa leu2-3 leu2-112 his3-1 u r d - 5 2 trpl-289 This DBY747 mrs2::HIS3 [rho+mit+] GW7/gd2-21.2/rhoo MATa leu2-3 leu2-112 his3-1 u r d - 5 2 trpl-289 This of [rho"] mrs2::HIS3 KGF177 MATa karl his4 trp5 ade6 [rho+mit-; S. uuarum] KGF177/rhoo MATa karl his4 trp5 ade6 [rho"] IC8/rhoo CK506 GF132-10A
Origin or Reference
ATCC 44774 (19) (19) study; disruption of study; EtdBr treatment GW7/gd2-21.2 G. Faye" (22) This study; EtdBr treatment of KGF177 (20) I. Bousquet' (14) G. Faye"
MATa karl leul [rho"] MATa karl leul [rho+mit+;AbI1,2;AaI5a,5b] MATa [rho+mit+; his1 met6 AbI1,2,3;AaI1,2,5a-cl GF167-7B MATa lys2 [rho'mit';A~bI,ACaI,Aw] G. Faye" (22) DBY747/CK MATa leu2-3 leu2-112 his3-1 u r d - 5 2 trpl-289 This study' [rho+mit+; AbI1,2;AaI5a,5b] MATa 11x2-3 leu2-112 his3-1 u r d - 5 2 trpl-289 This study' DBY747/SU [rho+mit+; AbI1,2,3;AaI1,4,5a,5b;Aw] DBY747/grII MATa leu2-3 leu2-112 his3-1 urd-52 trpl-289 This study' [rho'mit'; AbI1,2,3;AaI1,2,5a-c] DBY747/wo MATa leu2-3 leu2-112 his3-1 u r d - 5 2 trpl-289 This study' Awl [rho+mit+; AcbI, AxaI, GW7/gd2-21.2/CK MATa leu2-3 leu2-112 his3-1 u r d - 5 2 trpl-289 This study' mrs2::HIS3 [rho+mit+; rho+mit+; AbI1,2;AaI5a,5b] GW7/gd2-21.2/SU MATa leu2-3 leu2-112 his3-1 urd-52 trpl-289 This study' mrs2::HIS3 [rho+mit+; AbI1,2,3;AaI1,4,5a,5b;Awl GW7/gd2-21.2/grII MATa leu2-3 leu2-112 his3-1 u r d - 5 2 trpl-289 This study' mrs2::HIS3 [rho+mit+;AbI1,2,3;AaI1,2,5a-c] GW7/gd2-21.2/~0 MATa leu2-3 leu2-112 his3-1 u r d - 5 2 trpl-289 This study' mrs2::HIS3 [rho+mit+;AC~I,AC~I,AUI Mitochondria were introduced in respective strains via cytoduction by the use of karl strains (23). * G. Faye, Institut Curie, Centre Universitaire, Orsay, France. E I. Bousquet, Centre de GBnitique MolBculaire, CNRS, Gif-sur-Yvette, France.
E. coli strains were cultivated on LB or 2 X TY media (25). DNA Subcloning and Sequencing-Appropriate DNA restriction fragments were subcloned into Bluescript vectors and sequencing was done with the didesoxy chaintermination method using the T7 sequencing kit (Pharmacia LKB Biotechnology Inc.). Restriction enzyme sites used for subcloning are shown in Fig. 1.For sequencing of some larger fragments we used specific primers. Nucleotide sequences of both strands were determined. For DNA sequence analysis we used the MICROGENIE program (Beckman). The SWISSPROT Protein Sequence Library (release 18, May 1991) and the EMBL Nucleotide Sequence Library (release 27, May 1991) were searched by use of the FASTPprogram (26). strains were transTransformation of Yeast and E. coli-E.coli formed by the CaCl, procedure (25). Yeast transformation was done using the lithium acetate method (27). Southern Blot Analyses-DNA preparation from yeast was performed following the method described in Ref. 28, whereas blotting and hybridization was done as described in Ref. 25. Northern Blot Analyses-Northern blot analyses were done as described previously (21). Analyses of Mitochondrial Translation Products-Labeling of mitochondrial proteins was done essentially as described by Haid et al. (29) except that cells were cultivated in YPR medium and starved for methionine prior to labeling in SR-meth medium (synthetic medium with raffinose as carbon source, lacking methionine). Cells were labeled with [35S]methionine(Amersham Corp.; 25 pCi/ml culture)
in the presence of cycloheximide (100 pglml). Small scale isolation of mitochondria was done according to the method described by Needleman and Tzagoloff (30). Proteins were separated on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and after fluorography the dried gels were autoradiographed (31). Cytochrome Spectra-Cells were grown on YPR-plates for 2 days, suspended in water, and washed twice with water. After treatment of the cells with an excess of sodium dithionite insuspension, the pellets were applied to the window of a home-made cuvette and frozen in liquid nitrogen. Spectra were recorded against several layers of Parafilm in a Hitachi 150-20spectrophotometer. RESULTS
Multi-copy Suppression-In a previous communication we have reported on the isolation of three nuclear genes, MRSZ, MRS3, and MRS4 from a yeast gene library. When present on the high copy number vectorYEpl3, these three wild-type genes can suppressthe phenotypic effect exerted by the mitmutationM1301 (19). This mit- mutation is asinglebase pair deletion in domain 3 of the group I1 intron bI1,the first intron of the cytochrome b gene COB; inviuo it causesa complete block in the excision of this intron and, due to a
Control of Group II Intron Splicing
6965
-
from performing import assays of the MRSB protein into isolated mitochondria as has been successfully done with the HS. MRSB and MRS4 proteins (21). MRS? 14.0 The DNA sequence of the MRSB gene and the deduced amino acid sequence were compared with sequences of the S, A I A X H GI D available data libraries (EMBL and SWISSPROT). We did not find any convincing similarities of the MRSZ sequences with those in the libraries, nor did we detect any consensus HI 3 0.4 kb sequence motifs which might represent RNA, DNA, or nuFIG. 1. Restriction map of inserts MRS2/4.8(H21) and cleotide binding elements. MRS2/2.2. MRS2/4.8(H21) is a BarnHI fragment originally shown Effects of the MRS2 Gene Disruption on Mitochondrial RNA to have suppressoractivity (19); MRS2/2.2 is a subclone which exerts Splicing-In order to inactivate the chromosomal copy of the the suppressingactivitywhen present on a high copy number plasmid. The arrow represents the coding region as deduced from the DNA MRSB gene, an internal 0.5-kb SauI fragment of this gene was replaced by a 1.7-kb fragment containing the HIS3 gene sequence. The mrs2::HISBgene disruption was constructed byreplacing the 0.5-kb SauIfragmentbya 1.7-kb BamHIfragment (Fig. 1).The haploid strain DBY747 rho+ was transformed containing the yeast HIS3 gene(34). A, AccI; E, BamHI; D, DraI; G, with this construct following the one-step gene disruption BglIl; H, HindIIl; I, Saul; P, Pstl; Sp, selected Sau3A sites; X,X h l . method (39). Stable HIS' transformants were isolated, and the replacement of the wild-type MRSB copy bythe disrupted polarity phenomenon in COB RNA splicing, of all down- one was confirmed by Southern hybridization (datanot stream introns (32, 33). shown). The mrs2::HIS3 gene-disrupted strain (GW7/gd2By Northern blot analyses we could demonstrate that the 21.2) was unable to grow on non-fermentable substrates (cf. MRS2 gene product is in fact involved in mitochondrial RNA Fig. 6). The disruption thus causes a pet- phenotype. This splicing: in transformants of the mutant (strain DBY747/ indicates that the MRSB gene product is essential for the M1301) with the MRSSgene onthe multi-copy plasmid formation of a functional respiratory chain. YEp351 we could detect a band that corresponded to the To determine apossible effect of the MRSZ gene disruption mature transcript of the COB gene and which is not present on mitochondrial RNA splicing, we have performed a series in the same strain transformed with YEp351 alone (data not of Northern hybridization experiments using mtRNA of shown). strain GW7/gd2-21.2 which contains the disrupted MRSZ Subcloning and Expression of the MRS2 Gene-Subcloning gene. The patterns of the COB and COX1 transcripts of this of the MRSZ gene was done by partial digestion of the original strain are shown in Fig. 3 in parallel with those of the wild4.8-kb' BamHI insert (19) with the restriction endonuclease type strain DBY747 and the intron bI1 mutant DBY747/ S a d A and cloning of fragments with sizes of 1.5-4 kb into a M1301, which are isogenic with strain GW7/gd2-21.2 except YEp351 vector linearized with BamHI. The suppressing ac- for the MRSB alleles and the bI1 mutation. tivity could be allocated to a 2.2-kb fragment (Fig. 1).SupProbes specific for the COB exon B1 (including part of the pressor activity of this fragment was lost when the 0.5-kb leader) and for the COB intron bI1detect the mature mRNA SauI fragment was replaced by the HIS3 gene (Fig. 1). and theexcised bI1 RNA as themajor COB transcripts in the By use of the OFAGE technique (35) and the1.1-kb HindIII wild-type strain (Fig. 3). The strains with the nuclear MRSB restriction fragment as a hybridization probe we were able to gene disruption (GW7/gd2-21.2) or with the bI1 intron mulocate the MRSZ gene on chromosome XV. tation (DBY747/M1301) lack these mature RNAs. Instead, Northern blot analyses of poly(A)+ RNA revealed that they accumulate high molecular weight RNAs of similar sizes. MRS2 is weakly transcribed in vivo. A transcriptof about 1.6 These have been shown to comprise all COB exonand intron kb could be detected by hybridization with a 32P-labeled1.1- sequences, or all but the last COB intron (32). kb HindIII fragment of MRS2. Transcription of MRS2 apThe pleiotropic effect on intron excision may be due to a pears not to be regulated by carbon source (data not shown). primary block in theexcision of the group I1 intron bI1 which, Sequence Analyses of MRS2"The sequence of the 2.2-kb as asecondary effect, prevents the expression of maturases in subclone is shown in Fig. 2. It contains one long open reading downstream introns andhence their excision from pre-mRNA frame which can code for a protein of 470 amino acids with a (32, 40-42). Alternatively, disruption of MRS2 may equally calculated M, of 54,186. block excision of group I and group I1 introns. This latter The codon bias index (36) of the predicted MRS2 protein has been calculated to be 0.17 indicating that the gene is possibility was ruled out by the following experiment. Mitopoorly expressed. The protein appearsto be hydrophilic; about chondria of strain KGF177 (Saccharomyces uuarum) and of 24% of its amino acids are either positively or negatively strain CK506(Saccharomyces cerevisiae), containing only charged, and the calculated polarity index (37) is 46.2%. The group I introns b14 and b15 or introns b13,b14 and b15, predicted amino acid sequence indicates that the protein is respectively (22, 14), were combined by cytoduction with the MRS2 disrupted nuclear background (strain GW7/gd2-21.2). basic with an excess of 70 basic over 47 acidic residues. Many of the nuclear encoded proteins that aredirected into The cytoductants accumulate mature COB mRNA (data not mitochondria have been shown to contain leader sequences. shown). The same result was obtained with a mrs2:: strain shown These leaders, which are cutoff during import intomitochon- lacking all group I1 (and some of the group I) introns as in Fig. 5 . We conclude therefore that excision of the group I dria, are assumed to form amphiphilic helices, which are rich in arginine, leucine, and serine but do not contain lysine or introns b13, bI4, and b15 from the COB precursor is only little any acidic residues (38). The N-terminal part of the predicted effected by the MRSS disruption, whereas splicing of the MRSB protein may form such a leader; this and theobserva- group I1 intronbI1 is totally blocked ina mrd:: strain. tion that MRSZ plays a role in mitochondrial RNA splicing Additionally, we were not able to investigate the excision of indicate that MRSB is a mitochondrial protein. Poor in vitro the group I intron b12, since there was no strain available so translation efficiency of the MRS2 gene prevented us so far far that lacks only bI1; excision of bI1 is necessary for splicing of b12, because this stepis dependent on a functional maturase ' The abbreviation used is: kb, kilobase(s). (32). 1.0 hb
,M
x~2'2
Control of Group II Intron Splicing
6966
GATCGACCAGCAGCTTGTATACCTACATACTTACATATACACTCCTAACAGATTTTTTTTTGTATGTTGTTCTTTCCTCTGTGGTGGTCCAGTCTTTTGTCTCTTTTTTTTTTTCTTCTT
120
TTTTTTTTTTTTTTTTTTTTTTGCAGCATTATGATAGAACAATAGGGCTCAAGATCGCACCAAGGCTAACAGTAACGGTATACGCAATCGTAGTGAAAGTGATTTTCAATCAAGCATCTC
240
ATGAATCGGCGTCTCCTGGTACGTTCTATATCTTGTTTCCAACCTTTGTCGAGAATAACTTTTGGAAGACCAAACACGCCATTTCTTAGAAAGTATGCTGACACATCCACTGCTGCAAAC
360
M
N
R
R
L
L
V
R
S
I
S
C
F
O
P
L
S
R
I
T
F
G
R
P
N
T
P
F
L
R
K
Y
A
D
T
S
T
A
A
N 480
ACCAACAGCACCATATTGCGGAAACAGTTACTATCGTTGAAGCCCATTTCTGCCTCTGATTCACTGTTCATTTCGTGTACGGTATTCAATTCTAAGGGAAATATTATCTCAATGTCCGAG
T
N
S
T
I
L
R
K
~
L
L
S
L
K
P
I
S
A
S
D
S
L
F
I
S
C
T
V
F
N
S
K
G
N
I
I
S
M
S
E 600
AAGTTTCCTAAATGGTCCTTTTTAACTGAACATTCCCTTTTCCCCAGAGACCTGAGGAAAATAGATAACTCCTCTATTGATATTATTCCAACCATCATGTGTAAGCCAAACTGTATTGTC
K F P K W S F L T E H S L F P R D L R K I D N S S I D I I P T I M C K P N C I V 720
ATCAACTTATTGCATATCAAAGCTCTTATCGAACGAGATAAGGTCTACGTCTTCGATACAACAAACCCTTCCGCCGCTGCCAAACTGAGTGTACTTATGTATGACTTGGAGTCTAAGTTG I N L L H I K A L I E R D K V Y V F D T T N P S A A A K L S V L M Y D L E S K L
840
TCCTCCACCAAGAATAACTCTCAATTTTACGAGCATAGAGCCCTCGAGAGTATTTTCATCAACGTAATGAGCGCACTGGAAACAGATTTCAAGCTTCACTCACAAATCTGTATTCAAATC
S
S
T
K
N
N
S
O
F
Y
E
H
R
A
L
E
S
I
F
I
N
V
M
S
A
L
E
T
D
F
K
L
H
S
O
I
C
I
~
I 960
TTAAATGATCTGGAAAACGAGGTCAATAGACTTAAACTGCGGCATCTTTTAATTAAGTCCAAAGATCTTACGCTTTTTTACCAAAAAACTTTATTGATTAGAGATCTATTAGATGAACTA L N D L E N E V N R L K L R H L L I K S K D L T L F Y ~ K T L L I R D L L D E
L
TTAGAAAACGACGATGATTTAGCAAACATGTACTTGACAGTTAAGAAGTCTCCTAAGGACAATTTTTCGGACTTGGAAATGCTTATAGAGACGTACTACACCCAATGTGATGAATACGTT L E N D D D L A N M Y L T V K K S P K D N F S D L E M L I E T Y Y T ~ C D E Y V
1200
CAGCAATCAGAATCTTTGATTCAGGATATCAAATCTACTGAAGAAATTGTCAACATCATATTGGACGCAAATAGAAATTCCTTAATGTTGTTGGAGTTGAAAGTTACCATCTACACGTTG
~
O
S
E
S
L
I
~
D
I
K
S
T
E
E
I
V
N
I
I
L
D
A
N
R
N
S
L
M
L
L
E
L
K
V
T
I
Y
F
T
V
A
S
V
L
P
A
F
Y
G
M
N
L
K
N
F
I
E
E
S
E
W
G
F
T
S
V
A
V
F
S
I
V
S
A
L
CTAAAGCGAAGGCGGAAGTGGTGGAAATCAACCAAGCAGCGGTTGGGAGTGCTACTTTATGGCAGCAGCTACACTAATAAGGCTAACCTGTCGAATAATAAGATTAATAAAGGTTTTTCA L K R R R K W W K S T K ~ R L G V L L Y G S S Y T N K A N L S N N K I N K G F
AAGGTGAAGAAATTTAACATGGACAATGATATTAAGAACAAGCAGAACAGAGATATGATTTGGAAATGGTTGATAGAAGACAAGAAAAATTGAAAGGCGATTATCATGATAAACTATACA
V
K
K
F
N
M
D
N
D
I
K
N
K
~
N
R
D
M
I
W
K
W
L
I
E
D
K
K
L
Y 1440
ATCACCAAGAAAAATTTTAATTCTTTAAGATTCGTGACAAAGATGACCATGTATCCAAACTCACCCGCAAACTCCAGTGTGTATCCTAAAACATCAGCGTCTATTGCCCTGACAAATAAA I T K K N F N S L R F V T K M T M Y P N S P A N S S V Y P K T S A S I A L T N K
K
T
1320
GGATTCACAGTAGCATCTGTTCTCCCCGGCATTCTATGGTATGAATTTAAAGAATTTCATCGAGGAGAGTGAATGGGGGTTTACTTCAGTGGCGGTATTTTCTATTGTTTCTGCCCTTTAT
C
1080
1560 S
1680
N
ATATTATTAGCATAAACCTGTATATATACATATGTAAGAAAAAAGGGTATTTAAAACTCTTTTTAACAGCAAATCTTTTGTCCTTGTAAAAACATAAATAATTAAACTAAGACTAATTAT
1800
ACAATGACACTTTTTTATTAAGAGCAAAATAAACAAAAAGAACTCCTATGCATTATTTTTCGTTTTATTTTAACTTCTTAAATGGAAAGCTTCTCCTTGAACAAACTTTCCAAATCATCT
1920
CCTTGAACAAACTTTCCAAATCATCAACTGCATCCTTTATAGCGGCTGGTTTATCACCCATACCTTGGAAAACATTACCTTTACCACCAGCCTTACCGCCGATGATACTGGACACCTTTT 2040 TGGCAAGCGCACAACCATCAATACCCTTGGCTAAAGCAGCATTGGAGATGTAACAACCGTGAGCAACACGACCTTCAGGGTCATTACCTGCCAATAAATAGATTGACTTGTCTTTGACAG
2160
AATCATTGGATTTCATGTAGTTGATC
FIG. 2. Nucleotide sequence of the 2.2-kb fragment carrying the MRS2 gene. The sequence of the sense strand is shown. The deduced amino acid sequence of the ORF identified as MRS2 is given in the one-letter code below the nucleotide sequence.
In order to test whether excision of group I1 introns in the COXl transcript isalso affected, Northern hybridization experiments were performed with probes specific for exon A4 and for the threegroup I1 introns in the COXl gene, aI1, a12, and aI5c (Figs. 3 and 4). As shown in Fig. 3 (panel C) no mature mRNA can be detected in themrs2:: strain. Excision of introns a11 and aI5c is independent of the presence or absence of other introns whereas excision of intron a12 requires that its openreadingframe, possibly encoding a RNA maturase, is in frame with the upstream -rnRNP exons A1 and A2 (43). Therefore, we have chosen a strain lacking intron a11 (GW7/gd2-21.2/SU) to directly monitor excision or non-excision of intron a12. The transcript patterns of the mrs2:: strains lack excised group I1 intron RNAs (aI1, aI5c; Fig. 4, panels B and D )or Pr COB-I. Probe: b l l Probe- A4 show only traces of excised a12 RNA (Fig. 4C), whereas the FIG. 3. RNA splicing deficiency of MRS2-disrupted yeast corresponding bands are very dominant in the wild type. strains. RNAs prepared from strain DBY747/M1301 (middle lanes, M f 3 0 f ) ,from strains DBY747 carrying the wild-type MRS2 (left Instead, the disruption of MRS2 leads to accumulation of lanes, MRS2) and from strain GW7/gd2-21.2 carrying the disrupted high molecular weight transcripts hybridizing with all group MRS2 (right lanes,mrs2::) were hybridized with radioactivity labeled, I1 introns. Taken together the results presentedabove show invitro generated RNA probes complementary to a part of the that the MRS2 gene product is an essential factor for the untranslated leader and the first exon (COB-1, panelA), to the first excision of all four groupI1 introns from yeast mitochondrial intron ( b l f ,panel B ) of the mitochondrial COB transcript or to exon COB and COXlpre-mRNAs. 4 of the COXl gene (A4, panel C).Bands representing the mature The COXl transcript pattern of the mrs2::HISB strains, COB or COXl mRNAs or the excised intron bI1 are marked by however, differs from that of the bI1 mutant M1301 (Fig. 3, arrowheads. probe A4); this mutation does not directly affect the excision
1
Control of GroupSplicing 11Intron
A
D
C
A
pJ
6967 6
:.
Z E
--RNA
Probe COB-leader
-a12
-a15c J
Pr . bl1
Pr.: all
Pr.: a12
Pr.. a15c
FIG. 4. Selective effect of the MRSB disruption on group I1 intron splicing. Panels A , B, and D show patterns of RNAs prepared from mitochondria of strains DBY747 ( M R S 2 , left lanes) and GW7/ gd2-21.2 (mrs2::, right lanes) which both contain thelong COXl gene. Panel C presents pattern of RNAs prepared from strains containing the S. uuarum mtDNA (DBY747/SU, MRSZ, left lane; GW7/gd221.2/SU, mrs2::, right lane). Equal amounts of theseRNAs were loaded to the gels, blotted, and hybridized with in vitro transcribed radioactivity labeled RNAs complementary to intron bI1of the COB aI5c of the COXlgene (panels gene ( p a n e l A )and to introns aI1, aI2, B-D,respectively). Bands representing the excised introns bI1, aI1, aI2, or aI5c are marked by arrowheads. Note that strain GW7/gd221.2/SU bears mitochondria of S. uuarum which lack the upstream intron aI1; hence, accumulation of pre-RNAs containing intron a12 directly reflects reduced splicing efficiency of this intron and is not due to a polarity effect.
Probe A4
FIG.5. Deletion of group I1 introns in a mrs2::HIS3 background leads to formation of mature transcripts of COB and COXl genes. RNAs were prepared from mit- strainDBY7471M1301 (lanes 1 ), from mit+ strains containing all introns (long: DBY747, MRS2, lanes2, and GW7/gd2-21.2, mrs2::, lanes 3 ) , from strains lacking all group I1 and some group I introns (A group 11: DBY747/ grII, MRSZ, lanes 4, and GW7/gd2-21.2/grII, mrs2::, lanes 5) or from strains lacking all introns (w/o introns: DBY747/wo, MRS2, lanes 6, and GW7/gd2-21.2/wo, mrs2::, lanes 7). Transcripts were hybridized with radioactively labeled RNAs complementary to the untranslated leader and the first exon of COB (COB-leader, left panel) and with exon 4 of the COXl gene (A4, right panel). Mature transcripts are marked by arrowheads. M1301 \
rnrs2.:
4
MRS2
w f o introns
MRS2 “
of COXl introns but it exerts only the so-called “box” effect, an interplaybetween the intronsb14 and a14 (44). The COXl transcript patternof the mrs2::HIS3 strain canbe interpreted as containing various combinations of the three group I1 introns and the intron a14. This indicates that the other group I introns of the COXl pre-mRNA areexcised in the absence of the MRS2gene product. A polarity effect similar to thatof the non-excised intron bI1 in COB pre-mRNA may not be detected with the COXl pre-mRNA since the effect of the MRS2 disruption onexcision of the first two introns a11 and a12 is somewhat leaky and thus allows for sufficient expression of maturases encoded in the downstream introns. A more direct test for the effect of the MRS2 gene disruption on the excision of group I introns was performed with mitochondria lacking all group I1 introns but retaining some group I introns only. For this experiment mitochondria from strain GF132-10A (retaining groupI introns b14,b15,a13, a14) were introducedintothe nuclearbackground of the MRS2-disrupted strainGW7/gd2-21.2 via cytoduction. In Fig. 5 the effect of MRS2 disruption on processing of COB and COXlRNAsis comparedinvariousmitochondrial backgrounds. In mrs2:: strains containing the “long” version of mitochondrial DNA (bI1-b15; aI1-aI5c) no mature mRNAs of both genes can be detected. In contrast, in mrs2:: cells lacking all group I1 and some group I introns ( A group 11) mature mRNAs are clearly present, although in a slightly lower amount than in the correspondent wild type. Further predominantbandsinthis figure correspond to COB mRNA+bI5(panel A ) and COXl mRNA+aI4 (panel B). These bands, which are also present in the wild type lanes, together with some other weaker precursor bands occurring only in the mrs2:: background might indicate that disruption of MRSB has also some effect on group I intron splicing. In the A group I1 strain (containing only introns a13 and a14)
mrs? MRSZ Agroupll
FIG. 6. Pet- phenotype of MRSB disrupted strains. Cells were streaked on glycerol plates (YPG) and incubated for 4 days a t 30 “C. Strains and designations are as in Fig. 5.
one would expect only three COXl precursor bands. The A4 probe we used contains also 870 bases of intron a14, and this intronis highly homologous tothe COB intron b14 (1). Additional precursor bands in a blot hybridized with the A4 probe are caused by a crosshybridization between these two introns. When transcripts from mitochondria that lack all known group I and group I1 introns (22) were analyzed no difference was seen between those from a MRS2 (wild type, DBY747/ wo) and a MRS2 disrupted (GW7/gd2-21.2/~0) strain (Fig. 5). Functions of the MRS2 Gene Product aside from RNA Splicing-As shown above, deletion of group I1 introns or of all introns from mtDNA apparently cures MRS2-disrupted strains of their defect in RNA processing. If the only function of the MRS2gene product were to participatein splicing, one would expect that this intron deletion would also restore growth of MRS2 disrupted cells on a non-fermentable substrate. This was not the case; the MRS2 gene disruption caused the same pet- phenotype irrespective of the mitochondrial genotype (Fig. 6). This suggests that the MRSZ gene product has another essential function in mitochondria. A comparisonof cytochrome spectra of wild-type (DBY747/ wo; MRS2)and mrs2::HIS3 (GW7/gd2-21.2/wo; mrs2::) strains with intron-less mtDNAs reveals a striking absence of cytochrome a.a3 and a dramatic decrease of cytochrome b in the MRS2 disrupted cells compared with wild-type cells (Fig. 7); a cytochrome spectrum of the mrs2:: strain GW7/
Control of Group 11Intron Splicing
6968
DISCUSSION
550
600
Wavelength/nm FIG.7. Disruption of MRS2 gene leads to a deficiency of cytochromes in an intron-less strain. Cells of strain DBY747, GW7/gd2-21.2, and DBY747IM1301 (marked MRS, mrs::, and M1301, respectively) were grown in YPR medium and prepared for the recording of low temperature spectra as described under “Materials and Methods.” cyt b, cytochrome b; d,cytochrome a.a3.
I
1-
COX38 ATPase6
FIG. 8. MRSS-disrupted strains exhibit a standard pattern of mitochondrial translation products. Labeling and analysis of mitochondrial translation products are described under “Materials and Methods.” The positions of the mitochondrially synthesized proteins are indicated by arrows: VAR1, ribosomal protein; COXI, COX2, and COX3, subunits I, 11, and I11 of cytochrome c oxidase: ATPase 6, subunit 6 of mitochondrial oligomycin-sensitive ATPase. Strains anddesignations are as in Fig. 5.
gd2-21.2 containing all mitochondrial introns is identical to that of strain DBY747/M1301 (data notshown). However, it is not the absence of the mitochondrially made subunits of the cytochrome oxidase that cause this cytochrome a.a3 deficiency. As shown in Fig. 8, patterns of mitochondrial translation products are similar in MRS2 and mrs2::HISB strains lacking all introns whereas mrs2::HIS3 cells containing all introns lack apocytochrome b and subunit I of cytochrome oxidase, as expected from their lack of the respective mature mRNAs. The only effect which we can see is a reduced amount in the bands representing COX3 and subunit 6of the ATPase. However, this effect is also seen in MRS2 cells with the cobmutation M1301 and therefore may be a secondary effect of their respiratory deficiency.
The MRS2 gene was found during a search for nuclear genes that suppress a mitochondrial mutation in a group I1 intron when this gene waspresent in high copy number (19). This suppressor activity suggested that theMRSB gene product might have a function in the excision of group I1 (and possibly also group I) introns from mitochondrial premRNAs. Here we show that disruption of this gene indeed blocks the excision of all four known group I1 introns from the COB and COX1 transcripts and has little effect on the excision of some group I introns. The MRS2 gene product may have a weak effect on mitochondrial RNA splicing in general, but it is an essential factor only for the excision of group I1 introns. In this respect the MRS2 gene is unique among the many known genes that affect mitochondrial RNA splicing. One of these genes, CBP2, is necessary for excision of only one single intron (bI5) (45), whereas another one, MRS1, is an indispensible factor for two group I introns (bI3 and aI5b) (13, 14). Yeasts lacking the respective introns are unaffected by the disruption of these nuclear genes. Onthe otherhand, most of the nuclear genes involved in mitochondrial RNA splicing described so far have other functions besides their involvement in the splicing process. CYT18 in N. crmsa and NAM2 in yeast code for proteins with dual function; they serve as tRNA synthetases and in addition are essential factors for group I intron excision (16, 17); PET54 affects translation of the mitochondrially encoded COX3 gene and also splicing of aI5b (46); NAMl is a mitochondrial transcription factor and also seems to be involved in mitochondrial splicing (47, 48). The MSS116 gene, whichturned out encode to a RNA helicase type protein, and the MSS18 gene both have a second, still unknown function besides their action onmitochondrial RNA splicing (18,49). Apparently the involvement in mitochondrial RNA splicing is also not the only function of the MRSZ gene product. This notion rests on the fact that disruption of the MRS2 gene causes a pet- phenotype, even when combined with a mitochondrial genome lacking the four known group I1 introns or all known introns (cf. Fig. 6). Furthermore, the intron-less mitochondria appear to synthesize the standard setof major mitochondrial proteins and yet lack the cytochrome a.a3 spectral bands (cf. Figs. 7 and 8). This parallels a study on the MSS116 gene (18), which is involved in both group I and group I1 intron excision; its disruption also resulted in a petphenotype, irrespective of the presence or absence of the known intronsin mtDNA. Both findings could easily be explained by the assumption that not all introns in yeast mtDNA have been identified, but this seems unlikely since this genome has been sequenced almost completely. Alternatively the products of both genes MSS116 and MRS2 might be involved in other processing or modification events of mitochondrial transcripts. Both of these explanations attribute to the MRSZ gene product the primary (and only) function in RNA processing. However, this does not easily explain the fact that cytochrome a.a3 is absent and cytochrome b is reduced in a MRS2 disrupted strain lacking all introns although the major, mitochondrially encoded proteins are synthesized. These data rather hint at theinvolvement of the MRS2 gene product in the formation of functional cytochrome complexes. It may fulfill a basic function in mitochondrial biogenesis, e.g. in the assembly of cytochromes as it has also been suggested for genes COX10 and COX11 (50, 51) or it may be involved in the expression or theimport of some nuclear encoded subunits of the cytochrome complexes.
Control of Group II Intron Splicing We conclude from our observation that the MRS2 gene product might serve other functions besides RNA splicing and thus behave like several of the other nuclear genes involved in mitochondrial RNA splicing (12). Yet, from our results we cannot exclude that MRSZ affects respiration and splicing fairly indirect. We are currently investigating spontaneous suppressor mutations of MRSZ that restore growth on nonfermentable carbon sources. Preliminary resultsreveal the existence of a class of dominant nuclear suppressors that restore growth on glycerol of mrs2:: strains lacking allintronsin mtDNA, butnot ofmrs2:: strains harboring the full set of mitochondrial introns (long version).' Thus, the suppressor mutants are independent of the MRSZ gene product as far as basic functions in mitochondrial biogenesis are concerned. However group I1 intron splicing remains dependent on this product. We conclude therefore that the gene product of MRS2 is indeed involved in two different processes of mitochondrial biogenesis, directly or indirectly, and that the splicing deficiency is not simply the consequence of some yet unknown process also leading to respiratory deficiency. Acknowledgments-We gratefully acknowledge our colleagues Ernst Jarosch, Jan Kreike, Renei! Schroeder, and Uwe von Ahsen for stimulating discussions and comments on this paper. We would like to thank G. Faye and I. Bousquet for the gift of a series of yeast strains, H. Ruis for providing the facilities for recording of cytochrome spectra, and C. Wilson for critical reading of the manuscript.
REFERENCES 1. Michel, F., Jacquier, A., and Dujon, B. (1982) Biochimie 64,867881 2. Michel, F., and Dujon, B. (1983) EMBO J. 2,33-38 3. Cech, T. R. (1989) Gene (Amst.) 82,191-203 4. Michel, F., Umesono, K., and Ozeki, H. (1989) Gene (Amst.) 8 2 , 5-30 5. Michel, F., and Westhof, E. (1990) J. Mol. Biol. 216, 585-610 6. Belfort, M. (1991) Cell 6 4 , 9-11 7. Cech, T. R.,Zaug,A. J., and Grabowski, P. J. (1981) Cell 2 7 , 487-496 8. Schmelzer, C., and Schweyen, R. J. (1986) Cell 4 6 , 557-565 9. Van der Veen, R., Arnberg, A. C., and Grivell, L. (1986) EMBO J. 6,1079-1084 10. Jacquier, A. (1990) Trends Biochern. Sci. 1 5 , 351-354 11. Grivell, L. A. (1989) Eur. J. Biochern. 182,477-493 12. Lambowitz, A. M., and Perlman, P. S. (1990) Trends Biochem. Sci. 15,440-444 13. Kreike, J., Schulze, M., Ahne, A., and Lang, B. F. (1987) EMBO J. 6,2123-2129 14. Bousquet, I., Dujardin, G., Poyton, R. O., and Slonimski, P. P. (1990) Curr. Genet. 1 8 , 117-124 15. Gampel, A., Nishikimi, M., and Tzagoloff, A. (1989) Mol. Cell. Biol. 9,5424-5433 16. Akins, R. A., and Lambowitz, A. M. (1987) Cell 60, 331-345 17. Herbert, C. J., Labouesse, M., Dujardin, G., and Slonimski, P. P. (1988) EMBO J. 7,473-483 18. Shraphin, B., Simon, M., Boulet, A., and Faye, G. (1989) Nature 337,84-87
G. Wiesenberger, unpublished results.
6969
19. Koll, H., Schmidt, C., Wiesenberger, G., and Schmelzer, C. (1987) Curr. Genet.12,503-509 20. Schmidt, C., Sollner, T., and Schweyen, R. J. (1987) Mol. Gen. Genet. 210,145-152 21. Wiesenberger, G., Link, T. A., von Ahsen, U., Waldherr, M., and Schweyen, R. J. (1990) J. Mol. Biol. 217,23-37 22. SBraphin, B., Boulet, A., Simon, M., and Faye, G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,6810-6814 23. Conde, J., and Fink, G. R. (1976) Proc. Natl. Acad. Sei. U. S. A . 73,3651-3655 24. Hill. J. E.. Mvers. A. M., Koerner, T. J., and Tzaaoloff. . A. (1986) . Yeast 2; 163-167 25. Maniatis. T.. Fritsch. E. F.. and Sambrook. J. (1982) Molecular C l o n i 4 : A 'hborato'ry M ~ n u a lCold , Spring Harbor Laboratory, Cold Spring Harbor, NY 26. Pearson, W. R., and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. U. S. A . 85,2444-2448 27. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D.,Seidman, J. G., Smith, J. A., and Struhl, K. (1987) Current Protocols in Molecular Biology, Green Publishing Associates and WileyInterscience, NY 28. Winston, F., Chumley, F., and Fink, G. R. (1983) Methods Enzymol. 101,122-228 29. Haid, A., Schweyen, R. J., Bechmann, H., Kaudewitz, F., Solioz, M., and Schatz, G. (1979) Eur. J. Biochern. 94,451-464 30. Needleman, R. B., and Tzagoloff, A. (1975) Anal. Biochem. 6 4 , 545-549 31. Harlow, E., and Lane, D. (1988) Antibodies:A Laboratory Manual, pp. 635-657, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 32. Schmelzer, C., Haid, A., Grosch, G., Schweyen, R. J., and Kaudewitz, F. (1981) J.Biol. Chern. 2 5 6 , 7610-7619 33. Schmelzer, C., Schmidt, C., May, K., and Schweyen, R. J. (1983) EMBO J. 2, 2047-2052 34. Struhl, K. (1985) Nucleic Acids Res. 13,8587-8601 35. Carle, G. F., and Olsen, M. (1984) Nucleic Acids Res. 1 2 , 56475664 36. Bennetzen, J. L., and Hall, B.D. (1982) J. Biol. Chern. 2 5 7 , 3026-3031 37. Vanderkooi, G., and Capaldi, R.A. (1972) Ann. N . Y. Acad. Sci. 195,135-138 38. Von Heijne, G. (1986) EMBO J. 5 , 1335-1342 39. Rothstein, R. J. (1983) Methods Enzymol. 101,202-211 40. Lazowska, J., Jacq, C., and Slonimski, P. P. (1980) Cell 22,333348 41. Lazowska, L., Claisse, M., Gargouri, A., Kotylak, Z., Spyridakis, A,, and Slonimski, P. P. (1989) J. Mol. Biol. 2 0 5 , 275-289 42. Weiss-Brummer, B., Rodel, G., Schweyen, R. J., and Kaudewitz, F. (1982) Cell 29,527-536 43. Carignani, G., Groudinski, O., Frezza, D., Schiavon, E., Bergatino, E., and Slonimski, P. P. (1983) Cell 35. 733-742 44. Labouesse, M., Netter, P., and Schroeder, R. (1984) Eur. J. Biochern. 144, 85-93 45. McGraw, P., and Tzagoloff, A. (1983) J. Biol. Chern. 258,94599468 46. Valencik, M. L., Kloeckener-Gruissem, B., Poyton, R. O., and McEwen, J. E. (1989) EMBO J. 8,3899-3904 47. Ben Asher, E., Groudinsky, O., Dujardin, G., Altamura, N., Ker215, morgant, M., and Slonimski, P. P. (1989) Mol. Gen. Genet. 517-528 48. Lisowski, T.(1990) Mol. Gen. Genet. 2 2 0 , 186-190 49. Seraphin, B., Simon, M., and Faye, G. (1988) EMBO J.7 , 14551464 50. Nobrega, M. P., Nobrega, F. G., and Tzagoloff, A. (1990) J. Biol. Chem. 265. 14220-14226 51. Tzagoloff, A.,-Capitanio, N., Nobrega, M. P., and Gatti, D. (1990) EMBO J. 9,2759-2764 '
