Yeast mitochondrial leucyl-tRNA synthetase CP1 domain has functionally diverged to accommodate RNA splicing at expense of hydrolytic editing

Abstract

The yeast mitochondrial leucyl-tRNA synthetase (ymLeuRS) performs dual essential roles in group I intron splicing and protein synthesis. A specific LeuRS domain called CP1 is responsible for clearing noncognate amino acids that are misactivated during aminoacylation. The ymLeuRS CP1 domain also plays a critical role in splicing. Herein, the ymLeuRS CP1 domain was isolated from the full-length enzyme and was active in RNA splicing in vitro. Unlike its Escherichia coli LeuRS CP1 domain counterpart, it failed to significantly hydrolyze misaminoacylated tRNA(Leu). In addition and in stark contrast to the yeast domain, the editing-active E. coli LeuRS CP1 domain failed to recapitulate the splicing activity of the full-length E. coli enzyme. Although LeuRS-dependent splicing activity is rooted in an ancient adaptation for its aminoacylation activity, these results suggest that the ymLeuRS has functionally diverged to confer a robust splicing activity. This adaptation could have come at some expense to the protein's housekeeping role in aminoacylation and editing.

FIGURE 1.

Primary and tertiary structure of the LeuRS CP1 domain. A, shown is a homology model of yeast mitochondrial LeuRS (17). B, multiple sequence alignment of LeuRS CP1 domain and flanking regions containing both the N- and C-terminal β-strand linkers and extensions is shown. The CP1 domain, β-strand linkers, and β-strand extensions are shown in blue, red, and green, respectively. Bracketed numbers indicate the number of amino acids of a peptide insert. The E. coli LeuRS A293 residue (37, 38) is highlighted in orange, and the corresponding peptide indicated by an orange line. It is absent in the yeast mitochondrial LeuRS. Each of the yeast mitochondrial CP1 domain constructs is indicated at the top of the figure as follows: CP1 (Ile-260 to Glu-431), CP1-β (Trp-238 to Leu-442), and CP1-βext (Lys-232 to Tyr-452). The N- and C-terminal ends of the CP1-βext construct are also indicated in the homology model. Scm, S. cerevisiae mitochondrial; Ec, Escherichia coli; Mt, M. tuberculosis.

FIGURE 2.

Species-specific LeuRS CP1 domain-dependent in vitro splicing of the bI4Δ1168 precursor RNA. The processing of the bI4Δ1168 pre-RNA was evaluated with respect to substrate pre-RNA processing (A), fraction of excised bI4 intron formed (B), and fraction of ligated B4-B5 exons formed (C). Calculations were based on the intensity of the phosphorimaged bands for the substrate and products bI4 and B4-B5 as well as other alternate bands that emerged during the reaction. Splicing reactions incorporated 1 μm pre-RNA and 1 μm LeuRS and were initiated with 1 mm guanosine. ▴, ecCP1-βext; ♦, ymCP1-βext. Error bars for each time point are the result of each reaction repeated in triplicate.

FIGURE 3.

In vivo RNA splicing activity of full-length E. coli LeuRS. Complementation assays were performed using yeast null strains HM410 (with all 13 mitochondrial introns) and HM402 (intronless) (23). Complementation is indicated by darker colonies grown on glucose (Glu) media and by growth on glycerol (Gly) media. The arrow indicates that the full-length E. coli LeuRS supports growth of HM410 on Gly media. Both strains were transformed with the parent vector pQB153T or with plasmids expressing the wild type full-length ymLeuRS (pymLRST) or E. coli LeuRS (pKIRAN).

FIGURE 4.

Deacylation activity of isolated ymLeuRS CP1 domain. Deacylation reactions included ∼2 μm [³H]Ile-tRNA^Leu and 1 μm enzyme. ▴, wild type; ○, ymCP1-β; ♦, ymCP1-βext; ■, no enzyme. Error bars for each time point are the result of each reaction repeated in triplicate.

FIGURE 5.

LeuRS CP1 domain extension-dependent splicing activity. In vitro bI4Δ1168 pre-RNA splicing was determined with respect to processing of the substrate pre-RNA (A), fraction of the excised bI4 intron formed (B), and fraction of the ligated B4-B5 exons formed (C). Splicing reactions incorporated 1 μm pre-RNA and 1 μm LeuRS and were initiated with 1 mm guanosine. ■, ymCP1-β; ♦, ymCP1-βext. Error bars for each time point are the result of each reaction repeated in triplicate.

FIGURE 6.

The Trp-238 mutation in the WIG peptide abolishes splicing but maintains sufficient aminoacylation activity. A, complementation assays used yeast null strains HM410 and HM402 and is indicated by darker colonies grown on glucose (Glu) media and by growth on glycerol (Gly) media. Arrows highlight colonies expressing full-length W238A and W238C mutant ymLeuRSs support growth of HM402 but not of HM410 on Gly media. Both strains were transformed with the parent vector pQB153T or with plasmids expressing the full-length wild type (pymLRST) or W238A (pMPW238A), W238C (pMPW238C), W238F (pMPW238F), and W238Y (pMPW238Y) mutant ymLeuRSs. B, RNA amplification via RT-PCR of total cellular RNA from yeast null strain HM410 expressing full-length wild type and Trp-238 mutant ymLRSs yielded a 250 base pair (bp) amplified product representing the B4-B5 ligated exon product on a 1% agarose gel. C, shown is a Northern blot of 100 μg of total cellular RNA isolated from HM410 yeast cells expressing full-length wild type and Trp-238 mutant ymLeuRSs. Hybridization was carried out with a ³²P-labeled B4-B5 exon junction probe. D, in vitro aminoacylation activity of full-length wild type and Trp-238 mutant LeuRS is shown. Reactions included 4 μm transcribed ymtRNA^Leu, 1 μm enzyme, and 21 μm [³H]leucine (150 μCi/ml) and were initiated with 4 mm ATP. ■, wild type; ▾, W238A; △, W238C; ○, W238F. Error bars for each time point result from each reaction repeated in triplicate.

FIGURE 7.

In vitro splicing activity of isolated CP1 domain-based ymLeuRS Trp-238 mutants. Evaluation of in vitro bI4Δ1168 pre-RNA splicing with respect to processing of substrate pre-RNA (A), fraction of bI4 excised intron (B), and fraction of the ligated B4-B5 exons (C) is shown. Splicing reactions incorporated 1 μm concentrations of substrate pre-RNA and 1 μm LeuRS and were initiated with 1 mm guanosine. ▴, WT ymCP1-βext; ●, W238C ymCP1-βext. Error bars for each time point result from each reaction repeated in triplicate.