CBT1 Interacts Genetically With CBP1 and the Mitochondrially Encoded Cytochrome b Gene and Is Required to Stabilize the Mature Cytochrome b mRNA of Saccharomyces cerevisiae
Timothy P. Ellis, Melissa S. Schonauer, Carol L. Dieckmann

Abstract

Mutation of a CCG sequence in the 5′-untranslated region of the mitochondrially encoded cytochrome b mRNA in Saccharomyces cerevisiae results in destabilization of the message and respiratory deficiency of the mutant strain. This phenotype mimics that of a mutation in the nuclear CBP1 gene. Here it is shown that overexpression of the nuclear CBT1 gene, due to a transposon insertion in the 5′-untranslated region, rescues the respiratory defects resulting from mutating the CCG sequence to ACG. Overexpressing alleles of CBT1 are allelic to soc1, a previously isolated suppressor of cbp1ts-induced temperature sensitivity of respiratory growth. Quantitative primer extension analysis indicated that cbt1 null strains have defects in 5′-end processing of precursor cytochrome b mRNA to the mature form. Cbt1p is also required for stabilizing the mature cytochrome b mRNA after 5′ processing.

OXIDATIVE phosphorylation is carried out by enzyme complexes located in the inner mitochondrial membrane. The formation of these complexes requires the concerted participation of genes encoded in both the nucleus and the mitochondrion. The mitochondrial DNA of the yeast Saccharomyces cerevisiae encodes seven subunits of the various complexes directly involved in oxidative phosphorylation. The rest of the subunits are encoded in the nucleus. Nuclear gene products are also involved in many other processes of organellar biogenesis including transcription, stabilization, processing, and translation of mitochondrial RNAs. Other factors affect import of nuclear-encoded proteins into the mitochondrion and assembly of the respiratory complexes and the mitochondrial H+-translocating ATP synthase. The subset of nuclear genes required for respiratory functions is known as PET genes (Costanzo and Fox 1990; Tzagoloff and Dieckmann 1990; Dieckmann and Staples 1994; Grivell et al. 1999).

CBP1 is a PET gene required for stabilization of mitochondrial cytochrome b (COB) RNAs (Dieckmann et al. 1982, 1984a,b). COB mRNA is transcribed as a unit with the upstream tRNAglu (Bonitz et al. 1982; Christianson et al. 1983). Processing of tRNAglu from the initial transcript by RNase P and tRNA 3′ endonuclease (Hollingsworth and Martin 1986; Chen and Martin 1988) releases the tRNA and precursor COB mRNA, which has a 5′ end at −1098 (relative to +1 of the initiating AUG). The precursor RNAs are processed to mature mRNAs with 5′ ends at −954 or −955. In cbp1 mutant strains, tRNAglu levels are near wild type, whereas precursor COB mRNA is reduced to 25% of wild type and mature COB mRNA is undetectable (Mittelmeier and Dieckmann 1993; Chen and Dieckmann 1994).

Previous deletion studies delineated a 64-nucleotide region of the COB 5′-untranslated region (UTR) from −961 to −898 that is sufficient for Cbp1p-mediated stabilization of COB mRNA (Mittelmeier and Dieckmann 1993). This sufficiency element encompasses the mapped positions of the mature 5′ ends of COB mRNA at −955/−954. Within this otherwise AU-rich element, the CCG sequence from −944 to −942 is required for Cbp1p-mediated stabilization of the COB mRNA (Chen and Dieckmann 1997). Mutant strains with the central C changed to A are equivalent in phenotype to cbp1 nulls, while mutation of either flanking nucleotide results in a temperature-sensitive respiratory defect. Several suppressors of the temperature-sensitive ACG and CCU mutations have been isolated and characterized (Chen and Dieckmann 1997; Chen et al. 1999; Islas-Osuna et al. 2003). Recovery of a dominant suppressor in CBP1 that suppresses the ACG mutation supports the hypothesis that Cbp1 interacts with the CCG region of the COB 5′-UTR. Other suppressors include knockout mutations of pet127, which is involved in 5′-end maturation of several mitochondrial RNAs (Wiesenberger and Fox 1997), a mutation linked to DSS1, the nuclease component of the mitochondrial 3′ to 5′ degradosome (Dziembowski et al. 1998), and soc1, an unmapped suppressor of respiratory defects in a cbp1ts strain (Staples and Dieckmann 1994). Here we show that soc1 is allelic to overexpressing alleles of the gene CBT1.

A previous study showed that deletion of the CBT1 gene affects processing and stability of the COB mRNA (Rieger et al. 1997). Here we report that overexpression of CBT1 suppresses the temperature-sensitive phenotype of mutations in the 5′-UTR of COB mRNA and can also suppress a temperature-sensitive cbp1 mutation. We show by primer extension analysis that deletion of CBT1 leads to a decrease in 5′ processing of the COB mRNA precursor to the mature form. Northern blot analyses of strains lacking the precursor RNA 5′ extension show that Cbt1p is required for the stability of the mature mRNA after processing.

MATERIALS AND METHODS

Yeast strains and media:

S. cerevisiae strains and their genotypes are listed in Table 1. Media used were as follows: YEPD [1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose], YEPG [1% (w/v) yeast extract, 2% (w/v) peptone, 3% (v/v) glycerol], and WO [0.17% (w/v) yeast nitrogen base without amino acids or ammonium sulfate, 0.5% (w/v) ammonium sulfate, 2% (w/v) glucose, and other supplements depending on the auxotrophy of the strain]. Solid media contained 2% (w/v) agar.

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TABLE 1

Yeast strains used in this study

Disruption of CBT1:

The CBT1 gene was amplified from −146 to +956 (relative to +1 of the initiating ATG), using PCR with the oligonucleotides 5′ CBT1 and 3′ CBT1 (see Table 2), and ligated to plasmid pGEM-T Easy (Promega, Madison, WI). The HIS3 gene from plasmid pUC18/HIS3 was digested with EcoRI and XhoI and ligated to the XhoI and MfeI sites at +609 and +730 of the CBT1 reading frame. The HIS3 reading frame is in the opposite orientation to that of CBT1. This cbt1∷HIS3 construct was digested with NotI, the enzyme was heat inactivated at 65° for 30 min, and the digested DNA was transformed into the appropriate yeast strain as described (Elble 1992).

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TABLE 2

Oligonucleotide primers used in this study

Construction of YEp351-CBT1:

The CBT1 gene was amplified from −146 to +1035, using PCR with the oligonucleotides 5′ CBT1 and CBT1 + 1029BAMAS, which introduced a BamHI restriction site at the 3′ end of the PCR product (see Table 2). The PCR product was ligated to plasmid pGEM-T Easy (Promega) and sequenced to verify that there were no PCR-induced errors (Arizona Research Labs Sequencing Facility, University of Arizona). CBT1 was removed from pGEM-T Easy and ligated to the 2μ vector YEp351, using the restriction enzymes PstI and BamHI.

Isolation of genomic DNA:

Total yeast cellular DNA was isolated by a rapid glass-bead vortexing method as described (Hoffman and Winston 1987).

PCR amplifications:

PCR amplification of the CBT1 gene was carried out in 100-μl reactions containing 1 μl of yeast whole-cell DNA extract; 50 pmol of each of the relevant primers, dATP, dCTP, dGTP, and dTTP; and 2.5 units of Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis) in 1× PCR reaction buffer (10 mm Tris-HCl pH 8.3, 1.5 mm MgCl2, 50 mm KCl). Thirty cycles of amplification were performed as follows: denaturation, 30 sec (2.5 min in the first cycle) at 94°; annealing, 30 sec at 55°; and extension, 2 min at 72°. Amplification of the 5′-untranslated region of CBT1 was performed with Pfu Turbo (Stratagene, La Jolla, CA) as per manufacturer's instructions with an annealing temperature of 60° and an extension time of 18 min.

Southern blot analysis of CBT1 loci:

Ten micrograms of total yeast cellular DNA was digested with the EcoRI restriction enzyme and separated on a 1% agarose gel. The DNA was transferred to a Nytran membrane, which was probed with a random-priming-labeled fragment of CBT1 from position −146 to position +956.

Isolation of whole-cell RNA:

Total RNA was isolated from midlogarithmic cultures as described (Caponigro et al. 1993) after growth in liquid YEPG medium at the permissive temperature (for primer extension experiments) or in YEPD medium (for Northern blot).

Isolation of poly(A)+ RNA:

Poly(A)+ RNA was isolated using the batch protocol of the Oligotex mRNA minikit as described by the supplier (QIAGEN, Valencia, CA).

Northern analysis of poly(A)+ RNA:

Ten micrograms of poly(A)+ RNAs was separated on a horizontal 1.25% (w/v) agarose gel in 1× TB buffer (83 mm Tris base, 89 mm boric acid). The RNAs were transferred to a Nytran membrane (Schleicher & Schuell, Keene, NH) and probed in hybridization solution (6× SSC, 10× Denhardt's solution, 0.1% SDS, 50 μg/ml carrier DNA) at 50°. The CBT1 probe was 32P-end-labeled oligonucleotide CBT1 + 32AS (Table 2). The ACT1 probe was 32P-end-labeled oligonucleotide ACT1 probe (Table 2). Blots were analyzed on a Phosphorimager (Amersham, Arlington Heights, IL).

Isolation of mitochondria:

Mitochondria were isolated according to the method described (Faye et al. 1974), except that Zymolyase 20T (Seikagaku, Rockville, MD) was used instead of Glusulase to produce spheroplasts.

Isolation of mitochondrial RNA:

Mitochondrial pellets were resuspended in 10 mm Tris pH 8.0. An equal volume of phenol was added, followed by incubation on ice for 5 min. The samples were centrifuged at 13,000 × g for 5 min and the supernatant was removed. The phenol extraction was repeated and the RNA-containing supernatant was dialyzed against distilled and deionized water overnight.

Primer extension analysis of mitochondrial mRNAs:

Primer extension analyses were done as described (Islas-Osuna et al. 2002) except annealing of the oligonucleotide to the RNA was performed at 45°. Oligonucleotides used for these analyses are shown in Table 2.

Determination of cell division times:

Cells were grown on WO solid media, with selection for YEP351-CBT1 where appropriate, and then inoculated into YEPG liquid media. At set time points, absorbance measurements were taken with a Klett-Summerson colorimeter.

RESULTS

Suppressors of ACG, sup-a3, sup-a4, and sup-a5 have Ty1 insertions upstream of the CBT1 gene:

In previous studies, a CCG triplet at position −944 to −942 of the COB 5′-untranslated region (where the A nucleotide of the initiating ATG is designated +1) was shown to be required for Cbp1p-mediated stabilization of COB mRNA (Chen and Dieckmann 1997). Suppressors of the temperature-sensitive respiratory growth phenotype of strains with the CCG triplet mutated to CCU or ACG have been linked (Chen et al. 1999) to the PET127 and DSS1 genes, which encode products involved in mitochondrial RNA metabolism (Wiesenberger and Fox 1997; Dziembowski et al. 1998). However, the genes representing several suppressor linkage groups (III, IV, and V) were not characterized. As deletion of CBT1 was shown to affect the biogenesis of COB mRNA (Rieger et al. 1997), this gene appeared to be a good candidate for a locus described by one of the uncharacterized suppressor groups.

Our original hypothesis was that there would be loss-of-function mutations in the coding sequences of CBT1. Therefore, we planned to amplify the coding sequences of CBT1 by PCR and determine if any base changes could be observed. Surprisingly, attempts to amplify the CBT1 gene in the ACG suppressors sup-a3 (group III), sup-a4 (group III), and sup-a5 (group IV) by PCR using primers 5′ CBT1 (−146) and 3′ CBT1 (+956) proved unsuccessful, although the predicted 1101-bp product was seen in wild-type and other suppressor strains (data not shown). Using primers to amplify the intergenic region between the upstream threonyl tRNA and CBT1, products several kilobases larger than the 415-bp product seen in wild-type LL20 and the ACG strain were observed in sup-a3, sup-a4, and sup-a5 (Figure 1A). The largest of these bands was sequenced in sup-a3, sup-a4, and sup-a5, and each contained a full Ty1 element. A predominant PCR product in the sup-a5 strain was 300 bp larger than the wild-type product and upon sequencing it proved to contain only the long terminal repeat (LTR) of a Ty1 element. The vast majority of Ty1 insertions within the yeast genome occur near tRNAs or other genes transcribed by RNA polymerase III (Kim et al. 1998).

Figure 1.—

Analysis of the 5′-untranslated region of CBT1. (A) PCR of tRNAthr-CBT1 intergenic region in ACG suppressor and soc1 strains. PCR amplification was carried out on whole-cell DNA with the oligonucleotides CBT1 + 32AS and TRNA-THR and analyzed on a 1% agarose gel. Verification of labeled products was done by extraction of the product from the gel and DNA sequencing. LL20 is the parental wild-type strain; ACG contains the temperature-sensitive ACG mutation in the 5′-UTR of the cytochrome b gene; sup-a3, sup-a4, and sup-a5 are independently isolated strains containing nuclear mutations that suppress the temperature-sensitive phenotype of the ACG mutation. sup-u9 and sup-u10 are uncharacterized nuclear-encoded suppressors of the CCU mutation in the 5′-UTR of COB mRNA (Chen and Dieckmann 1997); RSY1 is a cbp1ts strain; RSY19 is a cbp1ts soc1 strain (Staples and Dieckmann 1994); and Tysoc1 is a cbp1ts soc1 neoR strain where neomycin resistance is linked to the soc1 allele (R. R. Staples and C. L. Dieckmann, unpublished data). (B) Southern analysis of CBT1 loci in ACG suppressor and soc1 strains. Ten micrograms of total cellular DNA was digested with EcoRI and separated on a 1% agarose gel. The DNA was transferred to a Nytran membrane and probed with a random-primed labeled CBT1 −146 to +956 fragment. Markers were a mixture of λ-phage cut with HindIII and ϕX174 cut with HaeIII and migration of appropriate fragments is shown. Strains are as described in (A).

Since sup-a5 has been previously linked to soc1, a suppressor of a cbp1ts allele, the intergenic region of two soc1 mutant strains was also amplified. RSY19 harbors the original spontaneous soc1-1 suppressor mutation (Staples and Dieckmann 1994). Tysoc1 was isolated in an attempt to map the soc1 locus with a Ty1-neomycin resistance insertion element (R. R. Staples and C. L. Dieckmann, unpublished observation). As shown in Figure 1A, both soc1 strains had large PCR products that when sequenced proved to include full Ty1 elements as well as the smaller band containing only an LTR insertion, whereas the cbp1ts strain RSY1 had a wild-type-sized PCR product.

To further investigate the arrangement of the DNA encompassing the Ty1 insertions, genomic DNA in the threonyl tRNA-CBT1 intergenic interval was analyzed by Southern blotting (Figure 1B). Total DNA isolated from wild-type strain LL20 yielded two EcoRI fragments when probed with a random-primed CBT1 fragment (−146 to +956), one of 1730 bp (5′ end of CBT1 ORF and 5′-untranslated region), and one of 2329 bp (3′ end of ORF and untranslated region). The wild-type strain LL20 and the cbp1ts strain RSY1, as well as sup-u9 and sup-u10 (suppressors of CCU), have fragments of these sizes. The sup-a3, sup-a4, and sup-a5 strains, as well as the two soc1 strains RSY19 and Tysoc1, have the 3′ ORF 2329-bp fragment. However, instead of the 5′ ORF 1730-bp fragment, two larger fragments of ∼4000 and 6500 bp were observed. No smaller band was observed in sup-a5, RSY1, or Tysoc1 as might have been expected from the smaller PCR product (Figure 1A). The PCR products containing only the LTR of Ty1 are likely amplified from a minor deletion product carried in the sup-a5, RSY1, and Tysoc1 strains.

Sequencing of the PCR-amplified 5′ region of CBT1 in the ACG-suppressor strains and the soc1 strains demonstrated that there were two distinct Ty1 insertion sites in these strains. Sup-a3 and sup-a4 have a Ty1 element inserted into position −124 (relative to the CBT1 initiating ATG) and have a duplicated 5-bp sequence, CTATC. Sup-a5 and the soc1 strains RSY19 and Tysoc1 contain Ty1 insertions at position −151 and have a duplicated GAACA sequence. In the original sorting of the suppressor strains into linkage groups (Chen et al. 1999), sup-a3 and sup-a4 were in group III, while sup-a5 was in group IV and was linked to soc1. Subsequently, a cross of sup-a3 to soc1 showed linkage in 39 tetrads analyzed (M. A. Islas-Osuna, unpublished data). Taken together, the analysis of the CBT1 upstream region and crosses to soc1 suggests that sup-a3, sup-a4, and sup-a5 are all in the soc1 linkage group.

Ty1 insertion in the 5′-UTR of CBT1 results in overexpression:

To determine whether the insertion of the Ty1 elements into the 5′-UTR had an effect on CBT1 expression, poly(A)+ RNA was analyzed by Northern blot (Figure 2). CBT1 mRNA levels in sup-a3, sup-a4, and sup-a5 were 19- to 25-fold higher than those in the wild-type LL20, as standardized to the amount of ACT1 mRNA in each strain.

Figure 2.—

Northern analysis of CBT1 mRNA levels in ACG suppressor strains. Ten micrograms of poly(A)+ mRNA was separated on a 1.25% agarose gel. The RNA was transferred to a Nytran membrane and probed with end-labeled oligonucleotides homologous to ACT1 (ACT1 probe) and CBT1 (CBT1 + 32AS). After normalization with the ACT1 control, the levels of CBT1 mRNA in each strain were quantified relative to LL20 as follows: LL20, 100%; sup-a3, 2500%; sup-a4, 2000%; sup-a5, 1900%; and ACG, 130%.

Plasmid-borne overexpression of CBT1 suppresses the temperature-sensitive phenotype of the ACG strain and a cbp1ts strain:

The Northern blot data suggested that overexpression of CBT1 is responsible for the rescue of the temperature-sensitive phenotype of the ACG strain. To test this idea, the ACG strain was transformed with the multicopy 2μ plasmid YEp351-CBT1. YEp351-CBT1 partially rescued the temperature-sensitive phenotype of the ACG strain (Table 3), while the control YEp351 plasmid did not. Likewise YEp351-CBT1 suppressed the temperature-sensitive defect of a cbp1ts strain (Table 3). Thus overexpression of CBT1 can rescue conditional mutations in either the COB mRNA or CBP1.

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TABLE 3

Generation times of ACG and cbp1ts strains rescued by plasmid-borne overexpression of CBT1

Deletion of CBT1 affects 5′ processing of COB mRNA:

The product of the CBP1 gene interacts with the 5′ end of COB mRNA (Chen and Dieckmann 1997). The finding that overexpression of CBT1 rescues mutations in either the 5′ end of COB mRNA or CBP1 infers that Cbt1p also acts on the 5′ end of the COB mRNA. To test this hypothesis, cbt1 was disrupted in LL20 and iLL20, which is isogenic to LL20 in the nucleus but contains an intronless mitochondrial genome, and the 5′ end of COB mRNA was analyzed by quantitative primer extension analysis. In wild type, the level of precursor COB mRNA is ∼10% of the level of mature COB mRNA (Chen et al. 1999) (Figure 3). Similar amounts of precursor and mature RNAs are in the cbt1 null strains and the total abundance of COB mRNA is lower (Figure 3). The processing defect is slightly greater in an intron-containing strain (Figure 3). Therefore, Cbt1p is involved in the 5′ processing of COB mRNA to the mature form. A strain containing the overexpression allele of CBT1 and wild-type mtDNA (sup-a3rho+) showed no effect on the COB mRNA levels, the ratio of mature to precursor RNAs, or growth on nonfermentable substrates (data not shown and Chen et al. 1999), suggesting that overexpression of CBT1 has a phenotype only when the Cbp1p-COB mRNA interaction is perturbed and that CBT1 is not a rate-limiting step in COB mRNA processing or stabilization.

Figure 3.—

Primer extension analysis of COB mRNAs in Δcbt1 strains. Quantitative primer extension analyses were done on total RNA essentially as described (Islas-Osuna et al. 2002) except that primer Cob5B + 3 was used in place of Cob6B. Pre-COB, precursor cytochrome b mRNA; Mat-COB, mature cytochrome b mRNA. COX2 was used as a loading control. Quantitation of RNAs in strains with mtDNA containing introns (LL20 and LL20Δcbt1) relative to the mature COB mRNA in LL20 is as follows: mature COB, LL20 100%, LL20Δcbt1 29%; precursor COB, LL20 16%, LL20Δcbt1 39%. Quantitation of RNAs in strains containing intronless mtDNA (iLL20 and iLL20Δcbt1) relative to the mature COB mRNA in iLL20 is as follows: mature COB, iLL20 100%, iLL20Δcbt1 34%; precursor COB, iLL20 8%, iLL20Δcbt1 29%.

Deletion of CBT1 results in accumulation of 15S and RPM1 RNAs with unprocessed 5′ ends:

The defect in 5′ processing of COB in the cbt1 deletion strains is similar to that previously shown for pet127 mutants (Wiesenberger and Fox 1997), although it is not as pronounced. No detectable mature COB mRNA is in pet127 strains, whereas some COB mRNA with mature 5′ ends is in the cbt1 deletion strains. pet127 mutants also have defects in the processing of other mitochondrial RNAs that require 5′ processing, including VAR1, the bicistronic ATP8/6 mRNA, 15S rRNA (Wiesenberger and Fox 1997), and RPM1, encoding the RNA component of RNase P (G. Wiesenberger, personal communication). To test whether deletions of CBT1 also caused processing defects in these RNAs, primer extension analysis was performed with primers specific for these RNAs (Figure 4). Primer extension of the LL20Δpet127 strain was included to show the positions of bands resulting from extension of precursor mRNAs (Wiesenberger and Fox 1997). Bands larger than the mature band are unprocessed, partially processed, or artifacts resulting from premature halting of the reverse transcriptase. However, as they are all larger than the mature RNA, the relative amounts of these bands demonstrate the presence or absence of a processing defect. There was no discernable difference between ATP8/6 mRNAs in a cbt1 null strain and a wild-type strain. There was a significant increase in the levels of VAR1 message in the cbt1 null strain compared to LL20 but no defect in 5′-end processing (Figure 4, Table 4). This increase in VAR1 mRNA levels is possibly the result of a compensatory mechanism that attempts to rescue respiration by increasing levels of Var1p, a mitochondrial ribosomal protein, and thus mitochondrial translation rates.

Figure 4.—

Primer extension of 15S rRNA, RPM1 RNA, VAR1 mRNA, and ATP8/6 mRNA in Δcbt1 strains. Quantitative primer extension analyses were done essentially as described (Islas-Osuna et al. 2002) except that mitochondrial RNA was used as the template and annealing was performed at 45°. Mat, mature form of RNA. Arrows represent RNAs that are not processed to the mature form. COX2 was used as a loading control. Quantitation of RNAs is shown in Table 4.

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TABLE 4

Quantitation of mitochondrial RNAs in a Δcbt1 strain

Deletion of CBT1 had a slight effect on the ratio of unprocessed to mature 15S rRNA and a more pronounced effect on processing of the RPM1 RNA (Figure 4, Table 4). Neither effect of Δcbt1 was as great as that observed in the pet127 strain. Interestingly, both the mature 15S and RPM1 RNAs in the pet127 null strain were shorter than that observed in either LL20 or LL20Δcbt1. These data suggest that processing to the shorter RNA in the pet127 mutant strain occurs via some other, normally minor, nuclease activity. Overexpression of CBT1 in a strain with wild-type mtDNA displayed no effect on the processing of any of the RNAs tested when compared to LL20 (data not shown).

Cbt1p is required to stabilize mature cytochrome b mRNA:

The TG955 strain contains a mitochondrial genome in which the sequence between the 3′-processing site of tRNAglu and the 5′ end of mature COB mRNA is deleted (Chen and Dieckmann 1994). Removal of tRNAglu from the multigenic precursor by the 3′ tRNA endonuclease results in the formation of a COB mRNA with a mature 5′ end at position −955 that requires no further processing. Deletion of CBP1 in this strain resulted in destabilization of the COB mRNA, demonstrating that Cbp1p is required for stabilizing the mature COB mRNA after processing (Chen and Dieckmann 1994). To determine whether Cbt1p is also required after processing for stabilization of the mature COB mRNA, CBT1 was deleted in the TG955 strain. Northern analysis showed that the double TG955 Δcbt1 mutant had lowered levels of COB mRNA similar to the Δcbt1 strain (Figure 5). We hypothesize that Cbt1p, like Cbp1p, is required to stabilize the mature COB mRNA.

Figure 5.—

Northern analysis of COB mRNA in a Δcbt1 strain with no precursor form of COB. Approximately 5 μg of mitochondrial RNA was loaded onto a 1.25% agarose gel, electrophoresed, blotted onto Nytran, and probed separately with the various probes. Blots were analyzed on a Phosphorimager. The ATP9 signal was used as a loading control for quantitation purposes.TG955 strains have cytochrome b mRNA precursor sequence deleted from −1098 to −956 (where the A of the initiating ATG is +1). Levels of COB mRNA were quantified relative to LL20 as follows: LL20, 100%; LL20Δcbt1, 16.4%; TG955, 116%; and TG955Δcbt1, 2%.

DISCUSSION

CBT1 is the third nuclear gene known to affect the 5′ processing and stabilization of mitochondrial COB RNAs. PET127 is required for processing of the 5′ end of the precursor RNA to the mature form. In pet127 strains, precursor RNA accumulates to levels equivalent to the combined level of precursor and mature RNAs in the wild-type strains, and there is no processing to the shorter mRNA. pet127 strains are also defective in 5′ processing of ATP8/6, VAR1 mRNAs, 15S rRNA (Wiesenberger and Fox 1997), and the mitochondrially encoded component of RNase P, RPM1 RNA (G. Wiesenberger, personal communication, and Figure 4). pet127 strains can respire except at high temperature (37°). CBP1 is required for the stabilization of COB RNAs (Dieckmann et al. 1982, 1984a,b; Mittelmeier and Dieckmann 1993; Chen and Dieckmann 1997). In cbp1 strains, the precursor COB mRNA, in which the 5′ end extends to the 3′ end of tRNAglu at position −1098 relative to the ATG at +1, is reduced to ∼10% of wild-type levels. The mature mRNA, in which the 5′-UTR extends to nucleotide −954 or −955, is undetectable. cbp1 strains cannot respire. Cbp1p likely interacts with the 5′-UTR of COB RNAs to protect them from degradation (Chen and Dieckmann 1997).

Deletion of CBT1 results in a decrease in the mature COB mRNA/precursor COB mRNA ratio (Figure 3). These data suggest that Cbt1p is required for efficient 5′ processing of COB precursor RNA. In the original report describing the characterization of CBT1 (Rieger et al. 1997), Northern analysis showed that the major COB mRNA species in cbt1 null strains is ∼200 nucleotides longer than the mature COB mRNA in wild-type strains. The authors attributed the increase in length to a 3′-end extension on the COB mRNA. The primer extension analysis presented here (Figure 3) shows that most of the COB mRNA in cbt1 null strains is unprocessed at the 5′ end and is 143 or 144 nucleotides longer than the mature form (Bonitz et al. 1982). These data suggest that the size difference observed in the Northern blots is due at least partially to a 5′-end processing defect, but do not exclude the possibility that cbt1 null strains have a defect in 3′-end processing as well. Mitochondrial 5′- and 3′-end processing factors have been shown to interact genetically. Overexpression of PET127, a 5′-processing factor (Wiesenberger and Fox 1997), can suppress deletions in SUV3 or DSS1, which encode the helicase and exonuclease components of the 3′ to 5′ mitochondrial degradosome (Wegierski et al. 1998). Additionally, suv3 or dss1 deletions result in accumulation of mRNAs with extensions at both the 3′ and 5′ ends (Dziembowski et al. 2003).

Here we show that CBT1 overexpression from Ty1-stimulated transcription or from 2μ plasmid transcripts suppresses a temperature-sensitive defect in the 5′-UTR of COB mRNA or a temperature-sensitive mutation in CBP1. The overexpressing suppressor cannot rescue a null CAG mutation in the 5′-UTR of COB (Chen et al. 1999), nor can it suppress a deletion of CBP1 (T. P. Ellis, unpublished observation). We also show that deletion of CBT1 lowers COB mRNA levels in strains carrying either a wild-type mitochondrial genome or a mitochondrial genome in which the region between the 3′ end of the tRNAglu and the mapped 5′ ends of the mature COB mRNAs is deleted (Figure 5). Together, these data suggest that, like Cbp1p, Cbt1p stabilizes COB after 5′-end processing. Cbt1p may protect cytochrome b mRNA via a direct interaction with Cbp1p and/or COB mRNA. When overexpressed in cbp1ts or ACG strains, Cbt1p could compensate for the partial loss of affinity between Cbp1p and COB mRNA. Such a parallel factor could be specific to COB mRNA or it could be a more general mitochondrial mRNA-stabilizing factor. In combination with a cbp1ts allele, Ty1-induced overexpression of CBT1 (soc1) resulted in a 1.5- to 3-fold increase in several of the different mitochondrial mRNAs (Staples and Dieckmann 1994). That study supports the idea that overexpression of Cbt1p has a more general stabilizing effect on several mitochondrial mRNAs.

Alternatively, Cbt1p may be part of a processing complex with Pet127p. Loss of Cbt1p affects processing of several RNAs, similar to Pet127p. However, unlike deletions of CBT1, Δpet127 suppresses the same COB or cbp1ts mutations that overexpression of CBT1 does. This may suggest that, if both proteins are part of the same complex, Pet127p is a destructive nuclease and Cbt1p confers specificity to the complex. When Pet127p is absent there is no processing of COB mRNAs and when Cbt1p is absent Pet127p is less active as a specific 5′-precursor-processing enzyme but is more destructive of RNA in general. When Cbt1p is overexpressed, Pet127p degradation activity is suppressed. How both the degradation activity of Pet127p and the stabilizing function of Cbt1p promote RNA processing will require additional experimentation.

Acknowledgments

We thank Michael Rice and Telsa Mittelmeier for critical reading of the manuscript. This research was supported by National Institutes of Health research grant GM34893 (to C.L.D.).

Footnotes

  • 1 Present address: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853.

  • Communicating editor: T. Stearns

  • Received September 17, 2004.
  • Accepted August 1, 2005.

References

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