X-ray crystallography shows how fungal tRNA ligase coordinates non-conventional splicing

Transfer RNAs (tRNAs) act as adaptor molecules that decode mRNA and deliver amino acids to ribosomes during protein synthesis in all living cells. During maturation, tRNAs undergo a series of processing and modification steps. Some tRNAs contain introns that must be enzymatically removed during their path to the mature form. This tRNA splicing process consists of two steps: (1) excision of the intron by a conserved splicing endonuclease, and (2) ligation of the resulting tRNA halves (exons) by a tRNA ligase (Figure 1a). All eukaryotes use the same conserved splicing endonuclease, but they have evolved distinct classes of tRNA ligase. Humans and other animals use one type, whereas fungi and plants rely on another [1]. In fungi, the tripartite tRNA ligase Trl1 joins the exons using three enzymatic modules within a single polypeptide (Figure 1b): an N-terminal ATP-dependent ligase (LIG), a central polynucleotide kinase (KIN), and a C-terminal cyclic phosphate diesterase (CPD). 

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Fig. 1: tRNA splicing by the fungal tRNA ligase Trl1. a) Schematic depiction of the two enzymatic steps during tRNA splicing. b) Domain organisation of Trl1. The ligase domain (LIG; both subdomains) from C. thermophilum Trl1 is shown in blue (PDB: 6N67), the polynucleotide kinase (KIN) in pink, and the cyclic phosphodiesterase (CPD) in green (PDB: 6U05). The linker region between the C. thermophilum Trl1 (CtTrl1) and C. albicans KIN-CPD (CaKIN-CPD) structures is indicated by a dashed line. 

Although the individual roles and structures of these domains have been described, their interplay during ligation and the principles of RNA substrate recognition have remained poorly understood, largely due to a lack of structural data. Given the divergence between human and fungal tRNA ligases, Trl1 is often discussed as a potential drug target for new antifungal therapies. These are urgently needed because of resistance in clinically relevant fungal pathogens and the associated health risks of invasive fungal infections. Moreover, RNA ligases are increasingly valuable tools in the expanding repertoire of RNA-sequencing technologies, which are instrumental in transcriptomics. The goal of this study was to understand how RNA ligases recognise and coordinate their RNA substrates. 

This research utilised the specific microfocus capabilities of ESRF beamline ID23-2 in combination with the automated “Mesh & Collect” multicrystal data-collection workflow [2]. Trl1-LIG was co-crystallised from the thermophilic fungus Chaetomium thermophilum with a tRNA-derived oligonucleotide. However, the resulting crystals were too small to collect complete diffraction data from a single crystal. Instead, more than a hundred micro-crystals were harvested into single sample holder loops. At the beamline, mesh scans identified crystal positions, and partial diffraction datasets were collected from each. After processing, the optimal combination of partial datasets was merged to yield the final dataset. This workflow enabled construction of a structural model of C. thermophilum Trl1-LIG in complex with RNA at 2.37 Å resolution, sufficient to provide mechanistic insights.

In the resulting crystal structure, the RNA substrate formed a duplex due to self-complementarity. Moreover, crystal-packing caused one ligase molecule to interact with four RNA strands arranged as two duplexes within the RNA binding groove (Figure 2a). However, the physiological substrates in the cell consist of two single-stranded RNA ends of the 5′ and 3′ exons. Thus, the challenge was to derive a physiologically relevant model for how Trl1-LIG coordinates the exons of a cleaved pre-tRNA from this crystal arrangement.

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Fig. 2: Structure of Trl1-LIG in complex with RNA. a) Surface representation of one C. thermophilum CtTrl1-LIG molecule showing the two RNA duplexes observed in the crystal lattice. The N-terminal domain is coloured blue and the C-terminal domain light blue. RNA strands (bronze and purple; sand and pink) are shown as cartoons, with the AMP cofactor in lime. b) Close-up of the active site with the activated AMP–RNA 5′ end; coordinating amino-acid residues are shown as sticks. The 2Fo–Fc electron-density OMIT map is displayed as a grey mesh. c) Surface representation of the CtTrl1-LIG (blue) model with bound exon halves of a tRNA anticodon stem-loop (pink and purple), derived from molecular-dynamics (MD) simulations.

One strand was found to be captured in the activated (adenylated) state, with an AMP moiety attached (Figure 2b). This on-pathway intermediate formed during crystallisation, thereby identifying and correctly positioning the 3′ exon. From this anchor point, the opposing end of the 5′ exon could be assigned based on spatial proximity and relative orientation. Biochemical assays combined with mutagenesis in Saccharomyces cerevisiae validated the assignment of the RNA strands in the crystal structure to their respective exon equivalents. 

Finally, computational simulations produced a model of Trl1-LIG in complex with a cleaved pre-tRNA substrate, consistent with the experimental data (Figure 2c). The simulations also suggested an important role of the unique C-terminal domain (CTD) of LIG in RNA binding by clamping the bound substrate. The CTD contains structural elements that guide the precise positioning of the exon ends, ensuring accurate ligation. The newly identified binding sites potentially provide additional target for Trl1-LIG-specific inhibitors in antifungal drug discovery. 

In summary, this study offers high-resolution structural insights into exon-exon coordination by RNA ligases. It identifies conserved principles of RNA-substrate recognition and highlights the mechanistic role of the CTD in substrate specificity. These findings deepen our understanding of RNA processing in eukaryotes and may open new avenues for antifungal therapy. Moreover, the growing importance of RNA ligases for sequencing technologies may benefit from these insights, enabling future enzyme engineering to improve performance and specificity.
 
Principal publication and authors
Structure of fungal tRNA ligase Trl1 with RNA reveals conserved substrate-binding principles, S. Köhler et al., Nat. Struct. Mol. Biol. (2025); https://doi.org/10.1038/s41594-025-01589-3
 

References
[1] J.L. Gerber, S. Köhler and J. Peschek, Biol. Chem. 403, 765-778 (2022).
[2] U. Zander et al., Acta Crystallogr D Biol Crystallogr. 71, 2328-2343 (2015).