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Mol Biol Cell. 2006 March; 17(3): 1184–1193.
doi: 10.1091/mbc.E05-08-0796.
PMCID: PMC1382308
Copyright © 2006, The American Society for Cell Biology
Balance between Transcription and RNA Degradation Is Vital for Saccharomyces cerevisiae Mitochondria: Reduced Transcription Rescues the Phenotype of Deficient RNA Degradation
Agata T. Rogowska,*† Olga Puchta,* Anna M. Czarnecka,* Aneta Kaniak,† Piotr P. Stepien,*† and Pawel Golik*†
* Department of Genetics, Warsaw University, 02-106 Warsaw, Poland
† Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland
Thomas Fox, Monitoring Editor
Address correspondence to: Pawel Golik (pgolik/at/ibb.waw.pl).
Received August 24, 2005; Accepted December 6, 2005.
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Abstract
The Saccharomyces cerevisiae SUV3 gene encodes the helicase component of the mitochondrial degradosome (mtEXO), the principal 3′-to-5′ exoribonuclease of yeast mitochondria responsible for RNA turnover and surveillance. Inactivation of SUV3 (suv3Δ) causes multiple defects related to overaccumulation of aberrant transcripts and precursors, leading to a disruption of mitochondrial gene expression and loss of respiratory function. We isolated spontaneous suppressors that partially restore mitochondrial function in suv3Δ strains devoid of mitochondrial introns and found that they correspond to partial loss-of-function mutations in genes encoding the two subunits of the mitochondrial RNA polymerase (Rpo41p and Mtf1p) that severely reduce the transcription rate in mitochondria. These results show that reducing the transcription rate rescues defects in RNA turnover and demonstrates directly the vital importance of maintaining the balance between RNA synthesis and degradation.
INTRODUCTION
The degradation of RNA is an essential element in the expression of genetic information. It is required to control RNA abundance and thus gene expression and to eliminate aberrant or defective molecules that inevitably form during RNA synthesis and maturation (RNA surveillance) (Vasudevan and Peltz, 2003 blue right-pointing triangle). The posttranscriptional mechanisms affecting mitochondrial gene expression, including RNA turnover, are of particular importance because transcriptional control is relatively simple and rudimentary. The single RNA polymerase (RNAP) of Saccharomyces cerevisiae mitochondria is composed of only two nuclear-encoded protein subunits—the core enzyme encoded by the RPO41 gene and a transcription initiation factor encoded by the MTF1 gene (Masters et al., 1987 blue right-pointing triangle; Jang and Jaehning, 1991 blue right-pointing triangle). The RNAP holoenzyme recognizes a simple nonanucleotide promoter sequence (Osinga et al., 1982 blue right-pointing triangle; Mangus et al., 1994 blue right-pointing triangle) and initiates synthesis of at least 13 primary multicistronic transcripts that undergo extensive processing to form mature RNAs (Christianson and Rabinowitz, 1983 blue right-pointing triangle; Tzagoloff and Myers, 1986 blue right-pointing triangle; Foury et al., 1998 blue right-pointing triangle; Gagliardi et al., 2004 blue right-pointing triangle; Schafer, 2005 blue right-pointing triangle). Such organization leaves little room for regulation at the transcription initiation level and makes posttranscriptional processes, including RNA degradation, key control points for mitochondrial gene expression.
The enzymes controlling RNA turnover in cells and organelles show great evolutionary divergence and are only partially conserved between mitochondria of different organisms (Gagliardi et al., 2004 blue right-pointing triangle). The enzymatic activity responsible for turnover is, however, on the basic level, similar in all the systems discovered so far—it is that of a 3′-to-5′ processive exoribonuclease, either hydrolytic or phosphorolytic. In most cases, the exoribonuclease activity is contained in a larger multiprotein complex that, in addition to exoribonucleases, also contains RNA helicases, and in certain cases, endonucleases. A model example of such a complex is the eubacterial degradosome (Carpousis, 2002 blue right-pointing triangle).
The first mitochondrial RNA degradation enzymatic complex was described in yeast S. cerevisiae and named mtEXO, or the mitochondrial degradosome (Margossian and Butow, 1996 blue right-pointing triangle; Dziembowski et al., 2003 blue right-pointing triangle). The mitochondrial degradosome is the main exoribonuclease in yeast mitochondria, which, unlike bacteria and animal or plant mitochondria, lack the phosphorolytic polynucleotide phosphorylase activity. Like the bacterial degradosome it contains an RNase and an RNA helicase; the subunit composition is, however, markedly different (Gagliardi et al., 2004 blue right-pointing triangle). The yeast mitochondrial degradosome is composed of only two protein subunits—an RNR (RNase II-like) superfamily exoribonuclease encoded by the DSS1/MSU1 (YMR287C) gene (Dmochowska et al., 1995 blue right-pointing triangle) and an NTP-dependent RNA helicase related to the DExH superfamily, encoded by the SUV3 (YPL029W) gene (Stepien et al., 1992 blue right-pointing triangle). The two proteins are tightly associated and the activity of both is essential for the functioning of the complex. The mitochondrial degradosome is capable of unwinding dsRNA regions and subsequently degrading single-stranded RNA in a 3′-to-5′ direction (Dziembowski et al., 2003 blue right-pointing triangle). The mtEXO complex interacts with the mitochondrial ribosome (Dziembowski et al., 2003 blue right-pointing triangle). Genetic interactions link the mitochondrial degradosome with the protein encoded by the PET127 gene, which is involved in 5′ processing and turnover of mitochondrial RNAs (Wiesenberger and Fox, 1997 blue right-pointing triangle; Wegierski et al., 1998 blue right-pointing triangle), and with the 5′ untranslated region of the CYTB mRNA (Chen et al., 1999 blue right-pointing triangle).
Inactivation of either the SUV3 or DSS1 gene gives a similar phenotype, corresponding to complete depletion of mitochondrial degradosome function. SUV3-deficient strains are all strictly respiratory deficient and rapidly lose wild-type mitochondrial DNA (mtDNA) converting to rho–/rho0 forms. Introduction of intronless mtDNA (Seraphin et al., 1987 blue right-pointing triangle) into the suv3Δ background (strain suv3Δ Δi) does not rescue the respiratory-deficient phenotype; however, it markedly improves the stability of such mtDNA to ~60% rho+ in an overnight YP-glucose culture (Dmochowska et al., 1995 blue right-pointing triangle; Golik et al., 1995 blue right-pointing triangle; Stepien et al., 1995 blue right-pointing triangle).
Among the plethora of molecular defects found in suv3Δ strains are overaccumulation of excised group I intronic sequences coupled with the destabilization of mature transcripts (Stepien et al., 1992 blue right-pointing triangle, 1995 blue right-pointing triangle; Golik et al., 1995 blue right-pointing triangle; Margossian et al., 1996 blue right-pointing triangle), accumulation of RNAs with abnormal 5′ and 3′ termini and of high-molecular-weight RNA precursors, variations in steady-state levels of mature transcripts, and disruption of mitochondrial translation (Dziembowski et al., 2003 blue right-pointing triangle). Together, these phenotypes suggest that the mtEXO complex is the primary activity responsible for RNA degradation and surveillance in yeast mitochondria.
A spontaneous suppressor allowing the suv3ΔΔi strain to grow on respiratory carbon sources was first described in the original suv3Δ deletion mutant (Dmochowska et al., 1995 blue right-pointing triangle; Golik et al., 1995 blue right-pointing triangle; Stepien et al., 1995 blue right-pointing triangle). The suppressor mutation, termed suB9, partially restored respiratory growth to the suv3Δ strain, as long as the mitochondrial genome did not contain the LSU-rRNA gene intron omega (Stepien et al., 1995 blue right-pointing triangle) or more than three introns in CYTB and COX1 genes (Golik et al., 1995 blue right-pointing triangle). The suB9 suppressor was found to be nuclear and monogenic, no further characterization was made at that time.
In this work, we show that mutations partially rescuing the phenotype associated with the loss of mitochondrial degradosome caused by disruption of the SUV3 gene, including the suB9 mutation, are point mutations in genes encoding subunits of the mitochondrial RNA polymerase that severely reduce the transcription rate. This is the first direct demonstration that maintaining the balance between RNA synthesis and degradation is crucial for the correct functioning of the mitochondrial genetic system.
MATERIALS AND METHODS
Strains, Media, and Classical Yeast Techniques
The S. cerevisiae strains used in this study are listed in Table 1. Standard yeast media and basic genetic methods were as described previously (Dujardin et al., 1980 blue right-pointing triangle; Burke et al., 2000 blue right-pointing triangle). The normal growth temperature was 30°C, and temperature sensitivity was tested at 36°C. The Singer MSM series 200 System micromanipulator (Singer Instruments, Watchet, Somerset, United Kingdom) was used for tetrad dissection and for the isolation of zygotes in isogenic crosses. Yeast were transformed using either the rapid or high-efficiency LiAc/SS-DNA/PEG protocol (Gietz and Woods, 2002 blue right-pointing triangle).
Table 1.
Table 1.
S. cerevisiae strains used in this study
Construction and Initial Characterization of suv3Δ Strains
The first suv3Δ mutants were constructed in the background of the BWG1 strain of S. cerevisiae (see Table 1 for all strain genotypes) as complete or partial deletions with the URA3 cassette (Stepien et al., 1992 blue right-pointing triangle). Because we planned to use such a strain as a starting point in the search for possible suppressors, we decided to recreate this system in the background of another, much better characterized strain W303, which has been extensively used in the study of nucleomitochondrial interactions. From a strain carrying the shorter (d2) deletion, we amplified the URA3 cassette with ~200 base pairs and ~100 base pairs SUV3 flanks at the 5′ and 3′ end, respectively, using primers 9090 and 9091. The PCR product was used to transform the W303/A/520 strain, which has the W303 nuclear background combined with the intronless mtDNA. The resulting strain, termed WSU0 Δi, behaved identically to the original suv3Δ strains, becoming respiratory deficient with a moderate decrease in the stability of the mitochondrial genome. To verify whether the partial nature of the deletion could have any influence on the phenotype, we also prepared an isogenic complete deletion strain by replacing the entire SUV3 open reading frame (ORF) with the KanMX4 selection module. We used DNA from the strain Y12799 originating from the Saccharomyces Genome Deletion Project (Winzeler et al., 1999 blue right-pointing triangle; Giaever et al., 2002 blue right-pointing triangle), which harbors the SUV3 deletion, as a template for PCR using the SUV3_A and SUV3_D primers. This gave a product containing the KanMX4 module with yeast genomic flanks upstream from the ATG and downstream from the termination codon of SUV3. Transformation of the W303/A/520 strain with this product gave a strain, termed, SUD1, carrying a deletion of the entire SUV3 ORF in the W303 background with the intronless mtDNA. In all our phenotypic analyses and in subsequent suppressor experiments, this strain behaved exactly like the partial deletion generated previously. We can therefore conclude that all the suv3Δ strains we constructed and used were functionally equivalent and corresponded to a complete loss of SUV3 gene function.
PCR Sequencing and Related Techniques
Yeast total DNA for PCR and plasmid isolation was prepared using the rapid phenol/glass beads protocol (Hoffman and Winston, 1987 blue right-pointing triangle). Primers 9090 (5′-AACTGCGGTTACATGGCCTA) and 9091 (5′-CTCGAAGATGAGAGGTGACC) were used to amplify the suv3::URA3 d2 disruption cassette (Stepien et al., 1992 blue right-pointing triangle). The suv3::KanMX deletion construct was amplified using primers SUV3_A (5′-TCAGAACACAATGTCCTTATTGAAA) and SUV3_D (5′-TATATTTTACTGCCCTTTGCTCAAC).
Primers MTF_A (5′-GATTATTGCGACTAATTTGAATGGT) and MTF_D (5′-CCTTTTCTTAAAGTTTTAGTTCCGC) were used to amplify the MTF1 gene, and primers MTF_C (5′-CAGTAGTAAGGGAGGCATTTACAGA), MTF_B (5′-AGTTCTTGTTTCCAATACAGGACAG), MTF350F (5′-TATTTGTTCCTGAAGTTCAAT), and MTF690R (5′-GTAGGCCATATTTCCGCAGCA) were used to sequence the MTF1 ORF.
Primers 976L (5′-CCACCAGCTTGTGAATAGGTT), 1764R (5′-CGAGCTTGTCGTTGAATGGA), 1724L (5′-ACTTCCATTCAACGACAAGC), 2427R (5′-CTCTCTTGCGGCTTCCGTTG), 2321L (5′-CACTGCCAACATTAGAGGAA), 3039R (5′-AGCTGGAGCTTCACCGTGAA), 2971L (5′-GTCGCTAAGGTATCTGTGCA), 3733R (5′-ACCACTTGAGACCAGAAGGT), 3714L (5′-ACCTTCTGGTCTCAAGTGGT), 4488R (5′-TAGTCCTAGTGGTGTTCGTCC), 4453L (5′-ATGTCATCCGTCATATGGAC), and 5210R (5′-TTGTAGTTCACGGCTCACGA) were used in pairs to amplify and sequence the RPO41 gene.
Vectors and Genomic Libraries
The wild-type S. cerevisiae genomic library in the pRS200 (ARS-CEN,TRP1) plasmid vector (Sikorski and Hieter, 1989 blue right-pointing triangle) constructed by P. Hieter was kindly donated by Dr. M. Johnston (Washington University, St. Louis, MO). YCplac111 (ARS-CEN, LEU2) and YCplac33 (ARS-CEN, URA3) (Gietz and Sugino, 1988 blue right-pointing triangle) were used as vectors. Plasmids pJJ1148 and pJJ1149 (Cliften et al., 2000 blue right-pointing triangle) containing the RPO41 gene cloned in the YCplac33 (ARS-CEN, URA3) and YCplac111 vectors, respectively, were kindly donated by Dr. Judith Jaehning (University of Colorado, Denver, CO).
RNA Preparation and Northern Hybridization
RNA was prepared from yeast mitochondria purified from log phase liquid cultures by differential centrifugation as described previously (Dziembowski et al., 2003 blue right-pointing triangle). RNA was run on a 0.8% agarose-formaldehyde gel and blotted onto nylon membrane as described previously (Tomecki et al., 2004 blue right-pointing triangle). Amount of mitochondrial protein (quantified by the Bradford assay) in the starting preparation was used to normalize the amount of RNA in each lane. Oligonucleotide probes 14S (5′-TATAAGCCCACCGCAGGTTCCCCTACGGTAACTGTA) and CYTB (5′-TATCTATGTATTAATTTAATTATATATTATTTATTAACTCTACCGAT) (Dziembowski et al., 2003 blue right-pointing triangle) were used to detect the 14S rRNA and CYTB mRNA, respectively.
In Organello Transcription Assay
Isolation of yeast mitochondria and run-on transcription in organello was performed essentially as described previously (Krause and Dieckmann, 2004 blue right-pointing triangle). Transcription reactions were performed in a buffer containing 50 mM HEPES-KOH, pH 8.0, 10 mM MgCl2, 25 mM KOAc, 10 mM dithiothreitol, 125 μM CTP, ATP, and GTP each, 50 μCi of [α-32P]UTP, 40 U of RiboLock RNase inhibitor (MBI Fermentas, Hanover, MD) and 5 μg of mitochondrial protein (as quantified by the Bradford assay) in a total volume of 50 μl. Two 2-μl aliquots were taken at each time point and spotted onto DEAE-cellulose filter discs (DE81; Whatman, Maidstone, United Kingdom). One of each pair of filters was washed four times in excess 0.4 M Na2HPO4 to remove unincorporated label, and the radioactivity was determined by liquid scintillation. The ratio of radioactive signal on the washed versus unwashed filter determined the incorporation for a given time point.
RESULTS
Spontaneous, Nuclear, Monogenic Suppressor Mutations Partially Restore Respiratory Competence in suv3Δ Strains Carrying the Intronless Mitochondrial Genome
In this work, we have undertaken a systematic study of spontaneous suppressors of the suv3Δ respiratory-deficient phenotype in the context of the intronless mitochondrial genome. Saturated liquid YPD cultures of the WSU0 Δi strain, described above, were plated on YPG plates at ~2.5 × 107 cells/plate and grown at 30°C for 7 d. Mitochondrial genome integrity was confirmed for each initial culture by crossing to the rho0 tester strain D273-10B/51. Colonies of respiratory-positive pseudorevertants were observed on each plate, with a median frequency of ~25 colonies/plate, which corresponds to an approximate mutation rate of 1 × 10–6 (Figure 1AFigure 1.). Both large and small pseudorevertant colonies were observed on each plate.
Figure 1.
Figure 1.
Figure 1.
Analysis of the suppressors of the suv3Δ phenotype. (A) Appearance of the [Gly+] pseudorevertant colonies in the intronless suv3Δ strain after 7 d of incubation on YPG at 30°C. (B) Selected pseudorevertant strains (numbered 1–6) (more ...)
Subsequent testing showed that all the pseudorevertant strains grew on respiratory media (glycerol) at normal temperature (30°C), albeit visibly slower than the wild-type W303/A/520 strain (Figure 1BFigure 1.). Interestingly, the respiratory-positive phenotype was totally lost from the pseudorevertant strains grown at a higher temperature (36°), which means that the suppression is a temperature-sensitive phenomenon (Figure 1CFigure 1.). Growth on YPD media was not affected at either temperature. Four of the pseudorevertant strains, chosen randomly from among both large and small colony classes, and named SUX1, SUX2, SUX3, and SUX4, were selected for further detailed analysis.
To verify whether the suppressor mutation was carried in the nuclear genome or in the mitochondrial DNA, the four selected pseudorevertant clones were converted to rho0 forms using ethidium bromide and fresh intronless mtDNA was introduced by cytoduction using the strain αKxNO41 (Seraphin et al., 1987 blue right-pointing triangle) as a donor. In all cases, the respiratory-positive phenotype was maintained in the cytoductants, suggesting that the suppressor mutations occurred in the nuclear genome.
Each pseudorevertant strain was then crossed to the SP55-11 strain (MATα, suv3Δ). The resulting diploids were homozygous for the suv3Δ deletion and heterozygous for the suppressor mutation. Each of the diploids failed to grow on respiratory media, indicating that the suppressor mutations were recessive.
To determine the number of different complementation groups among the suppressor mutations, the MATα suv3Δ su spores obtained by outcrossing the pseudorevertant strains to the wild-type isogenic strain (see next section) were crossed to each of the original pseudorevertant strains. Because the suppressor mutations are recessive, the resulting diploids would be respiratory competent only if both suppressor mutations occurred at the same locus. The results of this analysis indicated that there were at least two different complementation groups of the suppressor mutations, one (larger colonies, strains SUX1 and SUX2) corresponding to the original subB9 mutation, and the other (smaller colonies, SUX3 and SUX4) corresponding to another locus. They were given temporary names of su1 and su2, respectively. The strains carrying the su1 (SUX1) and su2 (SUX3) mutations were selected for further analysis. To summarize, the genetic analysis of the spontaneous suppressors of the suv3Δ disruption showed that the suppressors were nuclear, recessive, and monogenic and they occurred at two distinct loci in the genome. The recessive character of the suppressor mutations practically eliminated the conventional strategy of cloning the suppressor alleles based on their capability of restoring respiration to a suv3Δ strain.
The Suppressors of the suv3Δ Disruption Are Temperature-sensitive Partial Loss-of-Function Mutations in the Mitochondrial RNA Polymerase Genes RPO41 and MTF1
We crossed the pseudorevertant strains to the isogenic MATα wild-type W303-1B/520 strain. Diploids were isolated by micromanipulation and, following sporulation, tetrads were analyzed. This was essentially a two-point cross of suv3::URA3, su× SUV3+, SU+. The suv3Δ spores are readily identified by their [Ura+] phenotype, and the fact that the presence of the suppressor allele gives the suv3Δ spores ability to grow on glycerol at normal temperature facilitated the unambiguous assignment of each tetrad to one of the three classes. The ratio of PD:NPD:TT tetrads in each cross was close to 1:1:4, meaning that the suppressors were monogenic and not linked to the SUV3 locus or to the centromere.
In the NPD tetrads from the crosses described above, two spores carry the suv3Δ allele with the wild-type allele of the suppressor (SU1+ or SU2+) and have the [Ura+ Gly–] phenotype, whereas the other two are SUV3+ and carry the suppressor mutation. Two such NPD tetrads, DSW1-16 and DSW3-12, with the su1 and su2 mutations, respectively, were selected for further phenotypic analysis along with two TT tetrads DSW1-2 and DSW3-1. The spores were tested for uracil auxotrophy (as a marker of the suv3::URA3 disruption) and for growth on glucose (YPD) and glycerol (YPG) media both at normal and elevated (36°C) temperature. Presence of the suv3::URA3 disruption was additionally verified by PCR using primers 9090 and 9091. The results are shown in Figure 2Figure 2.. The suv3Δ, SU+ spores are [Ura+] and [Gly–] at both temperatures, as expected. The [Ura–] SUV3+, su spores are [Gly+] at the normal temperature, although their growth seems to be slower in comparison with a wild-type control. However, at the elevated temperature these spores become strictly [Gly–]. This means that both suppressor mutations give a temperature-sensitive respiratory-deficient phenotype in the context of the wild-type SUV3+ allele. In fact, the respiratory phenotype of these mutant strains is similar to that of the original pseudorevertant strain, regardless of the presence of the functional SUV3 allele. The loss of respiratory function at the elevated temperature is irreversible, mutant cultures grown overnight in liquid YPD at 36°C do not recover a [Gly+] phenotype upon subsequent transfer to 30°C, presumably because they lose mitochondrial DNA.
Figure 2.
Figure 2.
Figure 2.
Analysis of typical TT and NPD tetrads resulting from the cross of pseudorevertant strains SUX1 (su1 mutation) and SUX3 (su2 mutation) with an isogenic wild-type strain W303-1B/520. Tetrads DSW1-16 and DSW1-2 were selected as su1 NPD and su1 TT, respectively. (more ...)
This temperature-sensitive respiratory-deficient phenotype was used to design a screen for cloning of the suppressor genes. Strain DSW3–12/A was transformed with a wild-type S. cerevisiae genomic library in the pRS200 (ARS-CEN,TRP1) plasmid vector (see Materials and Methods). The [Trp+] transformants were screened for the ability to grow on YPG (glycerol) at 36°C. Ten such colonies were picked for further characterization and were all found to contain the same plasmid clone with an 8.4-kb insert corresponding to a fragment of chromosome XIII and covering two ORFs—RRP5 (YMR229C) and MTF1 (YMR228W). A 1.5-kb SacI-EcoRI fragment containing the MTF1 ORF and a 6.9-kb BamHI-EcoRI fragment containing the RRP5 ORF were subcloned into the YCplac111 (ARS-CEN, LEU2) plasmid vector to yield pMTF1 and pRRP5, respectively. Transformation of the DSW3-12/A strain with the pRRP5 plasmid did not restore respiratory growth at 36°C, whereas transformation with the pMTF1 plasmid restored wild-type respiratory growth at either temperature (Figure 3AFigure 3.). This indicates that the su2 mutation in DSW3-12/A occurred in the MTF1 gene, which encodes the transcription factor of the mitochondrial RNA polymerase (Schinkel et al., 1987 blue right-pointing triangle; Jang and Jaehning, 1991 blue right-pointing triangle). Temperature-sensitive respiratory-deficient mutants with defects in MTF1 had been previously described, along with similar mutations in the core mitochondrial RNA polymerase gene RPO41 (Shadel and Clayton, 1995 blue right-pointing triangle; Cliften et al., 1997 blue right-pointing triangle, 2000 blue right-pointing triangle; Karlok et al., 2002 blue right-pointing triangle; Matsunaga and Jaehning, 2004 blue right-pointing triangle). We decided therefore to verify directly whether the other suppressor mutation, su1, corresponded to the RPO41 gene. The DSW1-16/C strain, carrying the su1 mutation, was transformed with plasmids pJJ1148 or pJJ1149 (Cliften et al., 2000 blue right-pointing triangle) containing the wild-type RPO41 gene cloned in the YCplac33 (ARS-CEN, URA3) and YCplac111 vectors, respectively. All the obtained transformants were [Gly+] at both 30 and 36°C (Figure 3BFigure 3.), indicating that the su1 mutation occurred in the RPO41 gene. Passage of the DSW1-16/C/pJJ1148 transformant on fluoroorotic acid media led to the loss of respiratory growth at 36°C concomitant with the loss of uracil prototrophy, which further strengthened the evidence for the association of the su1 suppressor with a mutation in the RPO41 gene.
Figure 3.
Figure 3.
Figure 3.
Rescue of the wild-type (WT) respiratory growth on YPG at 36°C in SUV3+ strains carrying su1 and su2 mutations by low copy number plasmids carrying wild-type alleles of the respective genes. (A) Respiratory growth at 36°C in the SUV3+ (more ...)
To further verify the assignment of su1 and su2 suppressors to mutations in RPO41 and MTF1, we transformed the original pseudorevertant strains (suv3Δ, su) with plasmids pJJ1149 and pMTF1, carrying wild-type alleles of the respective genes. Because the suppressor alleles were shown to be recessive, introduction of the wild-type allele of the cognate gene should reverse the suppression and revert the transformant to respiratory deficiency at normal temperature. The transformants were tested on synthetic complete media (SC-leucine) with glycerol. The results (Figure 4Figure 4.) indicate that transforming the SUX1 strain with pJJ1149 (RPO41) and transforming the SUX3 strain with pMTF1 results in reversion of the suppressor phenotype, indicating that the plasmid-borne genes are indeed wild-type alleles of the suppressors. The original suppressor strain SDB9 (Stepien et al., 1995 blue right-pointing triangle) transformed with pJJ149 also reverts to the [Gly–] phenotype, which demonstrates that the suB9 mutation therein occurred in the RPO41 gene.
Figure 4.
Figure 4.
Figure 4.
Plasmid-borne wild-type alleles of MTF1 or RPO41 genes reverse the recessive suppression in suv3Δ pseudorevertant strains with su1 and su2 suppressor mutations. The suppressor phenotype was tested on synthetic complete (SC –leucine) media (more ...)
Because transformation of the pseudorevertant strain with plasmids carrying wild-type alleles of RPO41 or MTF1 is a quick way of determining the nature of suppression, we applied this strategy to a batch of eight additional suppressors isolated in the initial screen and assigned them to mutations in either RPO41 (5 colonies) or MTF1 (3 colonies). Similarly, spontaneous suppressors obtained in the SUD1 strain (carrying the KanMX4 deletion of the entire SUV3 ORF) were assigned to either of these two genes using this strategy.
This suggests that spontaneous [Gly+] suppressors arising in various suv3Δ strains carrying the intronless mitochondrial genome can be attributed to temperature-sensitive partial loss-of-function mutations in the RPO41 and MTF1 genes, encoding the two subunits of the mitochondrial RNA polymerase. The suppression occurs both with partial URA3 disruption and with total KanMX4 deletion alleles of suv3Δ
The Suppressor Mutations Are Novel Single Amino Acid Substitutions in RPO41 and MTF1
We selected the suppressor mutation in the MTF1 gene contained in the SUX3 strain, and two suppressors attributed to mutations in RPO41 (the SUX1 strain and the original SDB9 suppressor) for sequencing analysis. Fragments of genomic DNA corresponding to MTF1 and RPO41 genes were amplified and sequenced using primers described in Materials and Methods. Wild-type isogenic strains W303/A/520 and BWG1 were used as respective controls.
The SUX1 pseudorevertant strain was found to carry a single nucleotide substitution A1628G in the RPO41 ORF (all numbers start from the A in the initiation codon of the ORF), corresponding to the E543G substitution in the translated amino acid sequence. The SDB9 strain, compared with the parental strain BWG1, contained the G2932T substitution in the RPO41 gene, resulting in the V978F mutation in the amino acid sequence. Neither of these mutations has been previously described, they are both located in regions of the Rpo41p sequence displaying homology to the T3 and T7 phage RNA polymerases (Masters et al., 1987 blue right-pointing triangle).
The MTF1 gene in the SUX3 pseudorevertant, compared with the parental W303/A/520 strain contains a single mutation, C908T, resulting in a P303L substitution in the C-terminal region of the Mtf1p protein. The majority of the temperature-sensitive mutations in the MTF1 gene known so far were located within or close to the regions displaying homology to the σ family of bacterial transcription factors (Shadel and Clayton, 1995 blue right-pointing triangle; Cliften et al., 1997 blue right-pointing triangle). Deletion of the C-terminal domain, beyond residue 291, resulted in a temperature-sensitive petite phenotype (Shadel and Clayton, 1995 blue right-pointing triangle), corresponding to the effect of the P303L substitution observed in the DSW3-12/A spore.
Mutations in the RPO41 and MTF1 Genes Partially Suppressing the suv3Δ [Gly–] Phenotype Decrease the Transcription Rate of the Mitochondrial RNA Polymerase
Mutations in the mitochondrial RNA polymerase genes resulting in a similar, temperature-sensitive petite phenotype described previously (Shadel and Clayton, 1995 blue right-pointing triangle; Cliften et al., 1997 blue right-pointing triangle, 2000 blue right-pointing triangle; Karlok et al., 2002 blue right-pointing triangle; Matsunaga and Jaehning, 2004 blue right-pointing triangle) resulted in a general slowing of mitochondrial transcription and a decrease in the abundance of mature RNAs. As the phenotype of the suv3Δ mutation involves dysfunction of mitochondrial RNA degradation and turnover (Golik et al., 1995 blue right-pointing triangle; Stepien et al., 1995 blue right-pointing triangle; Dziembowski et al., 2003 blue right-pointing triangle), it was tempting to speculate that the suppressor mutations we discovered acted through lowering the transcription rate and reducing the RNA content in the organelle.
To verify this hypothesis, first we measured the steadystate levels of selected mitochondrial RNA transcripts by Northern hybridization. RNA obtained from purified mitochondria from the wild-type W303/A/520 (WT), suv3Δ (WSU0 Δi), su2 mutant in the MTF1 gene (DSW3-12/A), su1-2 mutant in the RPO41 gene (DSW1–16/C), and pseudorevertant suv3Δ su2 (SUX3) and suv3Δ su1-2 (SUX1) strains, grown at normal (30°C) and elevated (36°C) temperatures was separated on an agarose-formaldehyde gel and hybridized to 14S (SSU) rRNA and CYTB probes. The results are shown in Figure 5Figure 5.. As expected, the suv3Δ strain displays severe RNA processing abnormalities, resulting in very low levels of mature transcripts and accumulation of precursor species, including undefined high-molecular-weight smears as well as precursor bands. Strains carrying the suppressor mutations in MTF1 (su2) and RPO41 (su1-2) in the context of the wild-type SUV3 allele exhibit a marked decrease in the steady-state levels of 14S and CYTB mature transcripts (to ~33 and 20% of the wild-type level, respectively). In the pseudorevertant strains combining the suv3Δ deletion with the suppressor mutations, the steady-state levels of mature transcripts are restored to the levels similar to those observed in strains carrying the suppressor mutations alone, thus explaining how these mutations can partially restore respiratory competence in the suv3Δ background. Some accumulation of precursors is still observed, in particular in the suv3Δ su2 strain, but the proportion of mature transcript to precursor is much more favorable than in the suv3Δ strain.
Figure 5.
Figure 5.
Figure 5.
Northern blot analysis of steady-state levels of mitochondrial transcripts of the 14S rRNA and CYTB genes in wild-type W303/A/520 (WT), suv3Δ (WSU0 Δi), su2 mutant in the MTF1 gene (DSW3-12/A), su1-2 mutant in the RPO41 gene (DSW1-16/C), (more ...)
At the elevated temperature the two mitochondrial transcripts in all strains carrying the suppressor mutations were present at extremely low levels, while in the wild-type control their level was still normal. These results correlate with the previously observed phenotype of slower growth on respiratory substrates at normal temperature, and complete respiratory-deficiency at the elevated temperature, discussed above. Interestingly, the effect of the su2 mutation in the MTF1 gene on mitochondrial transcription is less pronounced than that of the su1 mutation in the RPO41 gene, which correlates with the increased accumulation of residual RNA precursors in the suv3Δ su2 strain compared with the suv3Δ su1 strain and slightly slower respiratory growth.
To verify whether the lower abundance of the mitochondrial transcripts observed in the suppressor mutant strains was indeed related to a decrease in transcription, we measured the mitochondrial transcription rates of the mutant strains using an in organello [α-32P]UTP incorporation assay (Krause and Dieckmann, 2004 blue right-pointing triangle). Mitochondria from wild-type W303/A/520 (WT), suv3Δ (WSU0 Δi), su2 mutant in the MTF1 gene (DSW3-12/A), su1-2 mutant in the RPO41 gene (DSW1-16/C), and pseudorevertant suv3Δ su2 (SUX3) and suv3Δ su1-2 (SUX1) strains, grown at a normal temperature to mid-log phase, were purified by differential centrifugation and incubated in transcription buffer containing [α-32P]UTP and an RNase inhibitor (see Materials and Methods). The results, shown in Figure 6Figure 6., demonstrate that although the wild-type strain shows typical rapid kinetics of the labeled precursor incorporation, the incorporation in the su mutant strains is much slower and never reaches wild-type levels, even upon prolonged incubation. Transcription in the suv3Δ strain is slightly lower than in the wild type, which may be related to the increased frequency of rho–/rho0 petites. Pseudorevertant strains suv3Δ su2 and suv3Δ su1-2 show slight improvement over the strains containing the suppressor mutations alone, which may be explained by the reduced RNA degradation activity in suv3Δ mitochondria. At 10 min of incubation, the incorporation in su1-2 (rpo41) and su2 (mtf1) mutants is ~10 and 18% of the wild-type value, respectively.
Figure 6.
Figure 6.
Figure 6.
In organello transcription assay measuring labeled [α-32P]UTP incorporation into purified mitochondria of the wild-type W303/A/520 (WT), suv3Δ (WSU0 Δi), su2 mutant in the MTF1 gene (DSW3-12/A), su1-2 mutant in the RPO41 gene (DSW1-16/C), (more ...)
We observed the same trend in several independent repetitions of the experiment, although absolute incorporation values were variable and the incorporation levels of the mutant strains were close to the sensitivity limit of the assay, which made a more rigorous statistical treatment of the quantitative results impossible. Positive incorporation in the mutant strains becomes more obvious when more than 5 μg mitochondrial preparation per reaction is used in the experiment; in such case, however, the wild-type preparation becomes overloaded and gives maximum signal already at the first time point.
These results demonstrate that the suppressor mutations in the RPO41 and MTF1 genes do indeed act by drastically slowing down the transcription rate in mitochondria, as demonstrated by the in organello incorporation assay. The effect on the steady-state level of mature RNAs seems slightly less pronounced, the two methods are, however, not directly comparable. Besides, more factors than just the transcription rate influence the steady-state level of transcripts in vivo which might explain the more moderate effect seen in the Northern hybridization assay. Although the effect of the suppressor mutations on transcription might seem drastic, respiratory competence of yeast mutants having <10% of the wild-type level of a mature mitochondrial transcript has been reported previously (Schmidt et al., 1998 blue right-pointing triangle). In the suv3Δ background, decreased transcription rates result in partial restoration of mature RNAs and decrease in the accumulation of precursors, so that the mature transcripts become the dominant form again.
DISCUSSION
The RNA helicase encoded by the SUV3 gene is a critical component of the yeast mitochondrial degradosome (Dziembowski et al., 2003 blue right-pointing triangle) and is essential for the expression and maintenance of the mitochondrial genome. A prominent feature of SUV3-deficient yeast strains is the accumulation of aberrant RNA species. Some of them can be attributed to unprocessed intronic sequences (Stepien et al., 1995 blue right-pointing triangle), but the abnormal accumulation of high-molecular-weight mitochondrial RNAs is also observed in the intronless strain (Dziembowski et al., 1998 blue right-pointing triangle, 2003 blue right-pointing triangle). Because some mature RNAs are still present in intronless mitochondria in degradosome-deficient strains, it has been suggested that impaired degradation of aberrant and unprocessed transcripts, rather than a block in the process of RNA maturation, is responsible for the molecular phenotype of SUV3 or DSS1/MSU1 deletion. In this model, the mitochondrial degradosome is responsible for the RNA surveillance function in the organelle.
The burden of aberrant and unprocessed RNA species could have a detrimental effect on the functioning of the mitochondrial genetic system. Accumulation of undegraded RNAs could disrupt the assembly of mitochondrial RNP complexes, and perturb the ribonucleotide pools. In particular, assembly and functioning of the mitochondrial ribosomes could be affected, because the accumulation of rRNA and VAR1 precursors has been observed in degradosome-deficient strains (Dziembowski et al., 1998 blue right-pointing triangle, 2003 blue right-pointing triangle). Perturbations in mitochondrial translation are known to affect the stability of mitochondrial DNA (Myers et al., 1985 blue right-pointing triangle), which would explain the elevated frequency of mitochondrial petites observed in suv3Δ strains (Stepien et al., 1992 blue right-pointing triangle, 1995 blue right-pointing triangle; Golik et al., 1995 blue right-pointing triangle). Defects in the quality control of ribosomal RNAs leading to defects in ribosome maturation were found to cause lethality in Escherichia coli strains devoid of the two major exoribonucleases, RNase R and polynucleotide phosphorylase (Cheng and Deutscher, 2003 blue right-pointing triangle). Although the polynucleotide phosphorylase has been found in human mitochondria (Piwowarski et al., 2003 blue right-pointing triangle), it is conspicuously absent from the mitochondria of S. cerevisiae. The mtEXO complex is therefore the sole major exoribonuclease of yeast mitochondria and its inactivation could cause disturbances similar in extent to those observed in bacterial cells lacking the two major RNase enzymes.
In this study, we demonstrated that spontaneous suppressor mutations partially rescuing the functioning of the mitochondrial genetic system in degradosome-deficient suv3Δ strains occur in the MTF1 (YMR228W) and RPO41 (YFL036W) genes encoding the two subunits of the mitochondrial RNA polymerase (Kelly et al., 1986 blue right-pointing triangle; Masters et al., 1987 blue right-pointing triangle; Jang and Jaehning, 1991 blue right-pointing triangle; Ulery and Jaehning, 1994 blue right-pointing triangle). They are temperature-sensitive, recessive, partial loss-of-function (hypomorphic) mutations that reduce the transcription rate at normal temperature and nearly abolish the mitochondrial RNA polymerase function at elevated temperature. At 30°C, mitochondrial transcription in mutant strains is <18% of wild-type level (as determined by nucleotide incorporation assay in organello), which also leads to reduced levels of mature transcripts (as detected by Northern blotting). It is also worth noting that the system we describe allows for positive selection of hypomorphic mutants in the RNA polymerase genes, which could be beneficial for studying the structure-function relationships in the RNAP protein subunits.
Partial rescue of the mitochondrial genetic function in the suppressor strains is linked to this decrease of mitochondrial transcription. Cells deficient in the mtEXO function cannot provide sufficient RNA degradation activity to cope with the production of unprocessed and aberrant RNAs associated with normal transcription rates. The pseudorevertant strains, with their reduced transcription rate, produce less RNA, including less unprocessed and/or aberrant transcripts, so that the mitochondrial genetic system can continue to function, albeit at a visibly lower level that translates to slower respiratory growth. In this model, decrease of the transcription rate caused by the suppressor mutations restores, at least partially, the balance between RNA synthesis and degradation disrupted by the genetic inactivation of the degradosome function. On the molecular level, this is manifested by shifting of the mature RNA/precursor ratios toward normal values, allowing sufficient expression of mitochondrially encoded genes. Although some overaccumulation of precursors is still observed in degradosome-deficient strains carrying the suppressor mutations, mature RNAs are the dominant form, like in wild-type mitochondria and unlike in the degradosome-deficient organelles.
It is possible that with the diminished transcriptional activity of suppressor mutants, the aberrant RNA species accumulate at a level that is so low that even without their removal, normally ensured by the degradosome, they do not interfere with mitochondrial function. Most likely, however, some residual exoribonuclease activity is responsible for the degradation of low levels of transcripts in suppressor strains. This activity could be provided by the RNase subunit of the mitochondrial degradosome encoded by the DSS1/MSU1 gene, probably in association with another RNA helicase. An obvious candidate would be the Mss1p mitochondrial RNA helicase (Seraphin et al., 1989 blue right-pointing triangle), which can partially rescue mitochondrial gene expression in suv3Δ strains when overexpressed from a multicopy plasmid (Minczuk et al., 2002 blue right-pointing triangle).
Another explanation would postulate the existence of another exoribonuclease in yeast mitochondria, which alone cannot provide sufficient degradation activity to provide the RNA turnover and surveillance function with the normal transcript levels produced by the wild-type RNA polymerase, but it can nevertheless cope with the severely reduced RNA quantities in the suppressor mutants. In E. coli, there are eight 3′-to-5′ exoribonucleases (Deutscher and Li, 2001 blue right-pointing triangle). Three of these, RNase II, RNase R, and polynucleotide phosphorylase, provide nonspecific processive activity required for the degradation of transcripts (Zuo and Deutscher, 2001 blue right-pointing triangle; Cheng and Deutscher, 2005 blue right-pointing triangle). The RNase component of the mitochondrial degradosome encoded by the DSS1/MSU1 gene remains, however, the only yeast processive 3′-to-5′ exoribonuclease found in mitochondria. The protein encoded by the NUC1 gene (Zassenhaus et al., 1988 blue right-pointing triangle), once thought to be the main mitochondrial nuclease, was found not to be involved in RNA degradation (Dziembowski et al., 1998 blue right-pointing triangle). The Ynt20p (Rex2p) 3′-to-5′ mitochondrial ribonuclease (Hanekamp and Thorsness, 1999 blue right-pointing triangle) has a distributive activity, similar to the bacterial oligoribonuclease (Zuo and Deutscher, 2001 blue right-pointing triangle) and is involved in the maturation of small RNAs (van Hoof et al., 2000 blue right-pointing triangle). The activity of Ynt20p family nucleases seems to be limited to degradation of very short single-stranded oligonucleotides (Nguyen et al., 2000 blue right-pointing triangle). None of the known proteins can be therefore considered as a candidate for the putative secondary 3′-5′ RNA degradation activity in yeast mitochondria that could supplement the mtEXO complex. Although this does not rule out the existence of such activity, no obvious candidate genes are apparent in the genome of S. cerevisiae. Alternatively, the 5′ end-directed degradation activity mediated by the product of the PET127 gene (Krause and Dieckmann, 2004 blue right-pointing triangle) could compensate for the lack of the 3′-to-5′ degradosome activity.
One of the particular characteristics of the mitochondrial genetic system is the close association between different levels of gene expression. RNA processing and translation have been shown to be physically coupled to transcription through a network of protein–protein and protein–RNA interactions (Rodeheffer et al., 2001 blue right-pointing triangle; Bryan et al., 2002 blue right-pointing triangle; Krause et al., 2004 blue right-pointing triangle). Because the mitochondrial degradosome is associated with the ribosome (Dziembowski et al., 2003 blue right-pointing triangle), it could also be a part of this network, offering another explanation for the genetic interaction between transcription and RNA degradation uncovered in this work.
The results of this study underscore the essential character of RNA degradation in the expression of genetic information. Mitochondria, with their simplified transcriptional control seem to be particularly dependent on posttranscriptional processes, including RNA degradation and surveillance. Our results clearly demonstrate that maintaining the balance between RNA synthesis and degradation is crucial to the functioning of the mitochondrial genetic system. Although the enzymatic machinery of mitochondrial gene expression shows considerable evolutionary divergence among phyla and is very different in higher eukaryotes in comparison with S. cerevisiae, a common theme of very simple transcription and complex posttranscriptional processing of RNAs is shared in mitochondria from yeasts to human. Understanding the mechanisms that maintain balance between the RNA synthesis and degradation is therefore a fundamental area of mitochondrial science.
Acknowledgments
We thank Dr. Judith Jaehning for the gift of plasmids pJJ1148 and pJJ1149 and Dr. Mark Johnston for the gift of the yeast plasmid library constructed by P. Hieter. We are grateful to Prof. Ewa Bartnik for critical reading of the manuscript. This work was supported by The State Committee for Scientific Research, KBN, through The Faculty of Biology, Warsaw University Intramural Grants BW#52/2003 and BW#19/2004.
Notes
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05–08–0796) on December 21, 2005.
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