Function of neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases

doi:10.1016/0092-8674(90)90119-Y

Cell

Volume 62, Issue 4, 24 August 1990, Pages 745-755

https://doi.org/10.1016/0092-8674(90)90119-Y Get rights and content

Abstract

Neurospora mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), which is encoded by nuclear gene cyt-18, functions in splicing group I introns. Analysis of intragenic partial revertants of the cyt-18-2 mutant and in vitro mutants of the cyt-18 protein expressed in E. coli showed that splicing activity of the cyt-18 protein is dependent on a small N-terminal domain that has no homolog in bacterial or yeast mt TyrRSs. This N-terminal splicing domain apparently acts together with other regions of the protein to promote splicing. Our findings support the hypothesis that idiosyncratic sequences in aminoacyl-tRNA synthetase may function in processes other than aminoacylation. Furthermore, they suggest that splicing activity of the Neurospora mt TyrRs was acquired after the divergence of Neurospora and yeast, and they demonstrate one mechanism whereby splicing factors may evolve from cellular RNA binding proteins.

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Cited by (69)

Structural divergence of the group I intron binding surface in fungal mitochondrial tyrosyl-tRNA synthetases that function in RNA splicing
2016, Journal of Biological Chemistry
Citation Excerpt :
These missing interactions between the three insertions together with the missing interactions between the insertions and protein core described in the preceding section suggest that the group I intron binding sites in both the An and Cp mtTyrRSs may be more flexible than in CYT-18, possibly to accommodate the splicing of different introns. The insertions in the catalytic domain of Pezizomycotina mtTyrRSs together with neighboring regions of the protein core form an extended group I intron binding surface on the side of the catalytic domain opposite the TyrRS active site (7, 12, 13, 26). The CYT-18 NTDs + Twort co-crystal structure shows that the protein interacts with both the P4-P6 and P3-P9 domains of the intron RNA, with most of the interactions occurring between the catalytic domain of one protein subunit (denoted subunit B) and the P4-P6 domain, particularly near its junction with the P3-P9 domain (12).
The mitochondrial tyrosyl-tRNA synthetases (mtTyrRSs) of Pezizomycotina fungi, a subphylum that includes many pathogenic species, are bifunctional proteins that both charge mitochondrial tRNA^Tyr and act as splicing cofactors for autocatalytic group I introns. Previous studies showed that one of these proteins, Neurospora crassa CYT-18, binds group I introns by using both its N-terminal catalytic and C-terminal anticodon binding domains and that the catalytic domain uses a newly evolved group I intron binding surface that includes an N-terminal extension and two small insertions (insertions 1 and 2) with distinctive features not found in non-splicing mtTyrRSs. To explore how this RNA binding surface diverged to accommodate different group I introns in other Pezizomycotina fungi, we determined x-ray crystal structures of C-terminally truncated Aspergillus nidulans and Coccidioides posadasii mtTyrRSs. Comparisons with previous N. crassa CYT-18 structures and a structural model of the Aspergillus fumigatus mtTyrRS showed that the overall topology of the group I intron binding surface is conserved but with variations in key intron binding regions, particularly the Pezizomycotina-specific insertions. These insertions, which arose by expansion of flexible termini or internal loops, show greater variation in structure and amino acids potentially involved in group I intron binding than do neighboring protein core regions, which also function in intron binding but may be more constrained to preserve mtTyrRS activity. Our results suggest a structural basis for the intron specificity of different Pezizomycotina mtTyrRSs, highlight flexible terminal and loop regions as major sites for enzyme diversification, and identify targets for therapeutic intervention by disrupting an essential RNA-protein interaction in pathogenic fungi.
Yeast mitochondrial leucyl-tRNA synthetase CP1 domain has functionally diverged to accommodate RNA splicing at expense of hydrolytic editing
2012, Journal of Biological Chemistry
Citation Excerpt :
Alternatively, they may possess distinct alternate mechanisms to suppress mistakes, such as precisely controlling mitochondrial amino acid concentrations or increased discrimination between cognate and noncognate amino acids. Interestingly and unlike TyrRS (CYT18), LeuRS has not acquired an idiosyncratic domain or peptide (34, 35) insert that is dedicated to RNA splicing. Thus, we hypothesize that evolutionary pressures on the ymLeuRS CP1 domain for dual functions in the cell compromised its post-transfer editing activity to facilitate its secondary but essential role of RNA splicing.
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.
Leucyl-tRNA synthetase-dependent and -independent activation of a Group I intron
2009, Journal of Biological Chemistry
Citation Excerpt :
In contrast, TyrRS does not have an editing domain. As discussed above, TyrRS enzymes that stimulate intron activity rely on small peptide insertions (30–34). These novel insertions form a new RNA binding surface for interactions with the final, folded form of the group I intron that are distinct from the tRNA binding site of TyrRS (34).
Leucyl-tRNA synthetase (LeuRS) is an essential RNA splicing factor for yeast mitochondrial introns. Intracellular experiments have suggested that it works in collaboration with a maturase that is encoded within the bI4 intron. RNA deletion mutants of the large bI4 intron were constructed to identify a competently folded intron for biochemical analysis. The minimized bI4 intron was active in RNA splicing and contrasts with previous proposals that the canonical core of the bI4 intron is deficient for catalysis. The activity of the minimized bI4 intron was enhanced in vitro by the presence of the bI4 maturase or LeuRS.
Crystal Structure of Human Mitochondrial Tyrosyl-tRNA Synthetase Reveals Common and Idiosyncratic Features
2007, Structure
Citation Excerpt :
Interestingly TyrRSs show large variations in sequence and length, due to insertions and appended domains (Wolf et al., 1999; Bedouelle, 2005). Additional functions result from these differences, like involvement in splicing for mitochondrial Neurospora crassa TyrRS (Cherniack et al., 1990) and cytokine activity for a fragment of human cytoplasmic TyrRS (Wakasugi and Schimmel, 1999). The present contribution focuses on human mitochondrial TyrRS (mt-TyrRS).
We report the structure of a strictly mitochondrial human synthetase, namely tyrosyl-tRNA synthetase (mt-TyrRS), in complex with an adenylate analog at 2.2 Å resolution. The structure is that of an active enzyme deprived of the C-terminal S4-like domain and resembles eubacterial TyrRSs with a canonical tyrosine-binding pocket and adenylate-binding residues typical of class I synthetases. Two bulges at the enzyme surface, not seen in eubacterial TyrRSs, correspond to conserved sequences in mt-TyrRSs. The synthetase electrostatic surface potential differs from that of other TyrRSs, including the human cytoplasmic homolog and the mitochondrial one from Neurospora crassa. The homodimeric human mt-TyrRS shows an asymmetry propagating from the dimer interface toward the two catalytic sites and extremities of each subunit. Mutagenesis of the catalytic domain reveals functional importance of Ser200 in line with an involvement of A73 rather than N1-N72 in tyrosine identity.
Evolution of the tRNA<sup>Tyr</sup>/TyrRS aminoacylation systems
2005, Biochimie
Citation Excerpt :
Particularly well studied is the mt-TyrRS from N. crassa (CYT-18 protein) that charges its cognate tRNATyr but also recognizes (but not aminoacylates) a shape in group I introns that mimic N. crassa tRNATyr including its large variable region [39]. Additional small idiosyncratic extensions at the N-terminus of mt-TyrRS plays also a key role in the splicing activity [38,77]. It was proposed that, in earlier time, group I introns were able to self-splice and became later dependent of protein CYT-18 that shared the needed prerequisites.
The tRNA identity rules ensuring fidelity of translation are globally conserved throughout evolution except for tyrosyl-tRNA synthetases (TyrRSs) that display species-specific tRNA recognition. This discrimination originates from the presence of a conserved identity pair, G1–C72, located at the top of the acceptor stem of tRNA^Tyr from eubacteria that is invariably replaced by an unusual C1–G72 pair in archæal and eubacterial tRNA^Tyr. In addition to the key role of pair 1–72 in tyrosylation, discriminator base A73, the anticodon triplet and the large variable region (present in eubacterial tRNA^Tyr but not found in eukaryal tRNA^Tyr) contribute to tyrosylation with variable strengths. Crystallographic structures of two tRNA^Tyr/TyrRS complexes revealed different interaction modes in accordance with the phylum-specificity. Recent functional studies on the human mitochondrial tRNA^Tyr/TyrRS system indicates strong deviations from the canonical tyrosylation rules. These differences are discussed in the light of the present knowledge on TyrRSs.
A tyrosyl-tRNA synthetase adapted to function in group I intron splicing by acquiring a new RNA binding surface
2005, Molecular Cell
We determined a 1.95 Å X-ray crystal structure of a C-terminally truncated Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) that functions in splicing group I introns. CYT-18's nucleotide binding fold and intermediate α-helical domains superimpose on those of bacterial TyrRSs, except for an N-terminal extension and two small insertions not found in nonsplicing bacterial enzymes. These additions surround the cyt-18-1 mutation site and are sites of suppressor mutations that restore splicing, but not synthetase activity. Highly constrained models based on directed hydroxyl radical cleavage assays show that the group I intron binds at a site formed in part by the three additions on the nucleotide binding fold surface opposite that which binds tRNA^Tyr. Our results show how essential proteins can progressively evolve new functions.

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^★: Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.

^†: Present address: Department of Biochemistry, Wayne State University Medical School, Detroit, Michigan 48201.

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Cell

ArticleFunction of neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases

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Article
Function of neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases