| Abstract |
The tRNA splicing endonuclease (TSEN) removes introns from precursor tRNAs (pre-tRNAs) in eukaryotes and archaea, but the architecture and subunit organization of plant TSEN remain poorly defined. Here, we integrated in planta bimolecular fluorescence complementation (BiFC) assays with AlphaFold3 (AF3)-based structural modeling, molecular dynamics simulations, molecular mechanics generalized Born surface area (MMGBSA) decomposition, and ConSurf analysis to characterize the Arabidopsis thaliana TSEN complex (AtTSEN), focusing on AtTSEN1, AtTSEN2, and AtTSEN54. Sequence- and structure-based homology searches did not detect a canonical TSEN15 homolog, a structural subunit conserved in yeast and human TSEN. BiFC supported pairwise interactions between AtTSEN54 and the catalytic subunits AtTSEN1 and AtTSEN2, but not between AtTSEN1 and AtTSEN2, suggesting AtTSEN54-mediated heterodimeric pairing. AF3 multimer modeling predicted a pseudo-symmetric archaeal-like (αβ)2 architecture for AtTSEN rather than a canonical eukaryotic αβγδ arrangement. In the AF3 pre-tRNA-bound model, AtTSEN54 was positioned near the pre-tRNA elbow, whereas the intron-containing anticodon arm was oriented asymmetrically toward the AtTSEN1 catalytic sites. Per-residue MMGBSA decomposition identified basic protein residues, particularly Arg and Lys, as putative pre-tRNA-binding hotspots, many of which corresponded to evolutionarily constrained interface positions across plant, fungal, and metazoan TSEN complexes. Together, these findings support an archaeal-like (αβ)2 structural model for AtTSEN and suggest that shared RNA-binding features are preserved alongside plant-specific remodeling of TSEN architecture.
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