Mitochondrial cysteinyl-tRNA synthetase is expressed via alternative transcriptional initiation regulated by energy metabolism in yeast cells

Abstract

Eukaryotes typically utilize two distinct aminoacyl-tRNA synthetase isoforms, one for cytosolic and one for mitochondrial protein synthesis. However, the genome of budding yeast (Saccharomyces cerevisiae) contains only one cysteinyl-tRNA synthetase gene (YNL247W, also known as CRS1). In this study, we report that CRS1 encodes both cytosolic and mitochondrial isoforms. The 5' complementary DNA end method and GFP reporter gene analyses indicated that yeast CRS1 expression yields two classes of mRNAs through alternative transcription starts: a long mRNA containing a mitochondrial targeting sequence and a short mRNA lacking this targeting sequence. We found that the mitochondrial Crs1 is the product of translation from the first initiation AUG codon on the long mRNA, whereas the cytosolic Crs1 is produced from the second in-frame AUG codon on the short mRNA. Genetic analysis and a ChIP assay revealed that the transcription factor heme activator protein (Hap) complex, which is involved in mitochondrial biogenesis, determines the transcription start sites of the CRS1 gene. We also noted that Hap complex-dependent initiation is regulated according to the needs of mitochondrial energy production. The results of our study indicate energy-dependent initiation of alternative transcription of CRS1 that results in production of two Crs1 isoforms, a finding that suggests Crs1's potential involvement in mitochondrial energy metabolism in yeast.

Keywords: Hap complex; alternative transcription; aminoacyl-tRNA synthetase; cysteinyl-tRNA synthetase; energy metabolism; gene regulation; mitochondrial bioenergetics; transcription; transcriptional start site; yeast.

Figure 1.

Dual localization of Crs1. A, fluorescence microscopy images of WT-GFP. The top schematic represents the genome structure of the WT-GFP strain used in this study. MitoTracker Red was used to stain the mitochondria. The arrowheads point to colocalization of the Crs1-GFP and mitochondrial signals. DIC, differential interference contrast; Mito, mitochondria. B, Western blot analysis of the whole (W), cytosolic (C), and mitochondrial (M) fractions. The cells were grown in YPGly to log phase, and then the purified mitochondrial fractions were isolated. Protein samples (5 μg) were prepared for detection of Crs1-GFP, Pgk1, and Por1. C, transcription start site analysis of CRS1. Multiple transcription start sites were determined via 5′-RACE analysis (n = 150). The x axis indicates the location of each mRNA start site and the y axis the number of times the sequence was found. The schematic at the top represents part of the CRS1 mRNA. The uppercase letters indicate the nucleotides of the transcription start sites, and the boxes indicate the nucleotides comprising the start codons. The translation start site in the database (RefSeq ID NM_001183085.1) is indicated by +1. D, localization analysis of Crs1 expressed with a nonoriginal promoter. WT cells harboring the pCRS1full-GFP or pCRS1ΔN13-GFP were found using fluorescence microscopy. pCRS1full-GFP is a plasmid that expresses Crs1 tagged with GFP at the C terminus plus the ADH1 promoter. pCRS1ΔN13-GFP is a plasmid that expresses the N-terminal truncated Crs1 tagged with GFP plus the ADH1 promoter. The schematic on the right represents the plasmid structures of pCRS1full-GFP and pCRS1ΔN13-GFP used here. MitoTracker Red was used to stain mitochondria. E, Western blot analysis of Crs1 expressed with a nonoriginal promoter. WT cells harboring pCRS1full-GFP or pCRS1ΔN13-GFP were grown in SG without uracil to log phase, and the whole, cytosolic, and mitochondrial fractions were isolated. Protein samples (10 μg) were prepared for detection of Crs1-GFP, Pgk1, and Por1.

Figure 2.

Analysis of an N-terminal truncated mutant of Crs1. A, fluorescence microscopy images of WT-GFP and ΔN13-GFP. Mito-ID Red was used for mitochondrial staining because MitoTracker Red cannot be applied to respiration-deficient mutants. The arrowheads indicate colocalization of the Crs1-GFP signal with the mitochondrial signal. The schematic on the right shows the genome structures of WT-GFP and the N-terminal truncated mutant (ΔN13-GFP). DIC, differential interference contrast; Mito, mitochondria. B, Western blot analysis of Crs1 localization in WT-GFP and ΔN13-GFP. WT-GFP and ΔN13-GFP were grown in SG to log phase, and the whole (W), cytosolic (C), and mitochondrial (M) fractions were isolated. Protein samples (10 μg) were prepared for detection of Crs1-GFP, Pgk1, and Por1. C, Western blot analysis of the cell extracts from WT-GFP and ΔN13-GFP. Protein samples (10 μg) were prepared for detection of Cox2, subunit II of cytochrome c oxidase (a mitochondrial genome-encoded gene), and Por1 (a nuclearly encoded gene). D, growth of WT-GFP and ΔN13-GFP in glucose or glycerol medium. Cells were grown in glucose medium and then spotted on glucose or glycerol medium.

Figure 3.

Hap complex–dependent regulation of transcription start sites. A, percentage of mitochondrial CRS1 mRNA for growth in glucose or glycerol medium. Cells were grown in glucose (YPD) or glycerol medium (YPGly) and harvested at log phase (L), early stationary phase (ES), and late stationary phase (LS) (Fig. S2). The percentage of mitochondrial CRS1 mRNA was determined using qPCR with total RNA. Results are presented as means ± S.D. (n = 5). Statistical significance was determined by one-way ANOVA with Tukey's test. *, p < 0.05 (versus the L result); #, p < 0.05 (versus the ES result). B, percentage of mitochondrial CRS1 mRNA in mitochondrial biogenesis–related gene mutants. WT, Δhap2, Δmig2, and Δrtg1 cells were grown in glucose medium (YPD) and harvested at L, ES, and LS. The percentage of mitochondrial CRS1 mRNA was determined using qPCR with total RNA. Results are presented as means ± S.D. (n = 3). Statistical significance was determined by one-way ANOVA with Tukey's test. *, p < 0.05 (versus the L result); #, p < 0.05 (versus the ES result). C, percentage of mitochondrial CRS1 mRNA in Hap complex–related gene mutants. WT, Δhap2, Δhap3, Δhap4, Δhap5, and Δhap-BS cells were grown in glucose medium and harvested at L, ES, and LS. The percentage of mitochondrial CRS1 mRNA was determined using qPCR with total RNA. Results are presented as means ± S.D. (n = 5). Statistical significance was determined by one-way ANOVA with Tukey's test. *, p < 0.05 (versus the L result); #, p < 0.05 (versus the ES result). D, percentage of mitochondrial CRS1 mRNA in HAP4-overexpressing cells. WT harboring the pAG426GPD-ccdB or pAG426GPD-HAP4 were grown in glucose medium (SC medium without uracil) and harvested at L, ES, and LS. The percentage of mitochondrial CRS1 mRNA was determined using qPCR with total RNA. Results are presented as means ± S.D. (n = 3). Statistical significance was determined by one-way ANOVA with Tukey's test. *, p < 0.05 (versus the L result); #, p < 0.05 (versus the ES result). E, ChIP with the CRS1 promoter. Cells with the HA tag inserted into the 3′ site of HAP2 in the genome were grown in glucose (YPD) or glycerol (YPGly) medium and harvested at L and LS. Binding of Hap2 to the promoter of CRS1 was determined by means of a ChIP assay with HA antibody. Input represents PCR results before immunoprecipitation (IP). F, percentage of mitochondrial CRS1 mRNA in RSC2 mutants. WT and Δrsc2 cells were grown in glucose medium (YPD) and harvested at L, ES, and LS. The percentage of mitochondrial CRS1 mRNA was determined using qPCR with total RNA. Results are presented as means ± S.D. (n = 5). Statistical significance was determined by one-way ANOVA with Tukey's test. *, p < 0.05 (versus the L result); #, p < 0.05 (versus the ES result). G, growth of WT-GFP, ΔN13-GFP, Δhap2, Δhap-BS, and Δrsc2 in glucose (YPD) or glycerol (YPGly) medium. Cells were grown in glucose medium and spotted on glucose or glycerol medium.

Figure 4.

Dual targeting mechanism of Crs1. CRS1 is transcribed from a single gene with different transcription start sites, which leads to the creation of mRNAs of different lengths: long and short. The long mRNA harboring the first upstream AUG is translated to the Crs1 protein with the MTS, which transports Crs1 to mitochondria. The short mRNA lacking the first AUG is translated from the second AUG to the Crs1 protein without the MTS. This type of Crs1 remains in the cytosol. The need for a specific mitochondrial function governs whether the Hap complex binds to the CCAAT sequence on the CRS1 promoter, which regulates the expression of the long (mitochondrial) mRNA.